Bioengineering strategies for gene delivery

C H A P T E R

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Bioengineering strategies for gene delivery Shahin Shams, Eduardo A. Silva Department of Biomedical Engineering, University of California, Davis, Davis, CA, United States

1 Introduction From deciphering DNA’s structure to the sequencing of the entire human genome, it is possible to argue that nucleic acids have been in the front-line of some of the most impactful scientific discoveries of the last century. More recently, methods of gene introduction, regulation, and editing have been the focus of promising therapeutic mechanisms in biomedical and pharmacological research endeavors. The formal definition of gene therapy, as defined by the National Institutes of Health (NIH), is the use of genes to treat or prevent disease [1]. Gene therapy can take many forms, such as replacing a mutated gene with a healthy copy, inactivating a mutated gene, or introducing a new gene into the body to help fight disease [1]. Gene therapies introduce foreign nucleic acid sequences to cells, which then can interact with the cell’s transcriptional and translational machinery to produce delivered nucleic acids or proteins. Due to the broad spectrum of potential disease targets and the ever-growing libraries of modular genes cassettes documented, gene therapies have been continuously investigated for treatment applications for decades. Historically, the first gene therapy clinical trial started in 1989, but early devastating complications regarding the safety of gene therapies slowed progress in the field [2]. These past failures motivated the need for better gene therapy designs for clinical application [2]. Several factors were addressed, ranging from the design of vectors themselves to the methods of their delivery to control spatiotemporal presence of therapeutic gene cassettes in target cell populations [3]. After over 2500 clinical trials and

Copyright © 2020 Tiago G. Fernandes, Maria Margarida Diogo & Joaquim M. S. Cabral. Published by Elsevier Inc. All rights reserved. https://doi.org/10.1016/B978-0-12-816221-7.00004-5

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almost 30 years, the first five gene therapies were approved by the Food and Drug Administration (FDA) between the years 2015 and 2019 [2]. Gene therapies present many features, such as localizing therapeutic levels of biomolecules, that make them desirable when compared to traditional systemic therapeutic options for tissue regeneration and disease treatment. For example, many therapeutic strategies employ the use of a bolus dose of protein that is systemically administered via oral ingestion, vascular infusion, or interstitial injections [4–6]. However, therapy via protein formulation presents many biological challenges including inherent difficulty in maintaining localized concentrations of the therapeutic dose due to the short half-life of the protein of interest, elimination of the protein to levels below therapeutic ranges by metabolic action, and off-target effects of the protein in nontargeted tissues [4, 7]. Alternatively, by introducing a gene rather than the protein itself, continuous localized production of the protein is possible that bypasses some of the above-­ mentioned limitations and challenges. In addition, upstream genetic elements and posttranscriptional regulatory mechanisms can be utilized for spatiotemporal controlled protein expression [8]. In general terms, gene therapies can be introduced via either direct or indirect strategies (Fig. 4.1). The direct application approach introduces the gene therapy vector in vivo. While direct applications are minimally invasive and comparatively simple, there are important challenges regarding the biosafety of this method that need to be considered. For example, localizing the therapeutic gene to the region of interest using direct application is difficult due to mass transport effects in tissue, therefore, not only does this method increase the uncertainty of dose but also jeopardizes nontargeted tissues by exposing them to the chance of unintentional transduction or transfection events [7]. In order to decrease the chances of off-target tissues being affected, vectors must be designed for conditional target cell interaction. However, even with vector design considerations, dosage may still be compromised by the movement of the vector down its concentration gradient into adjacent tissues, or systemically by gaining access to the bloodstream. In addition, upon therapeutic application, a potentially severe host immune response to the introduced vector could be possible [8]. In contrast, the indirect approach introduces the gene therapy vector outside the body, for example, it could involve the isolation of target cells from the patient and then genetically manipulating in the lab (i.e., ex vivo) before reintroduction to the patient. In other words, cells engineered by the indirect approach are therefore transfected or transduced in  vitro, cultured in the laboratory, and finally administered back to the patient. This strategy of retrieving the cells from a patient and manipulating them ex vivo is favored over the direct approach since it limits genetic modification to the cells in culture—minimizing the chance of off-target genetic manipulation. However, this method also presents several limitations and



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FIG.  4.1  Administration routes of gene therapies. The direct gene therapy strategy introduces genetic vectors in vivo, while the indirect gene therapy strategy manipulates cells ex vivo before reintroducing them to the body. Both direct and indirect gene therapy strategies can be incorporated with biomaterial carriers for the controlled delivery of vectors or cells, respectively.

challenges. For instance, isolating the cells of interest limits the cell types able to be manipulated ex vivo for gene therapy application [4]. In addition, this process is quite intensive from a research standpoint, requiring transfection or transduction processes to be precisely timed and extensive culturing of modified cells [7]. Due to the high technical complexity of cell isolation and manipulation, the costs associated with this approach are associated in kind. Localization of the manipulated cells upon reapplication to the patient also poses a problem similar to direct gene therapy approaches. The genetic activity of manipulated cells outside the target area may cause unintended effects in off-target tissues, jeopardizing said tissues while decreasing the intended activity of the gene therapy in the region of interest. In addition, the survival rate of the cells reintroduced tend to be low, also decreasing the effectiveness of the therapy [4]. In order to increase localization, viability, and efficiencies of both these therapeutic applications, engineering strategies have been employed. One possible engineering strategy is the combination of genetic vectors with material systems. Both naturally occurring and synthetic polymeric material platforms provide a promising approach for broad therapeutic

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applications of gene delivery. This wide range of material options makes it possible to select specific features that address the goals of the therapy of interest. In addition, many materials present extensive tunability that allows for precise control over biotransport kinetics, biochemical interactions, and strength—thus a single material platform can potentially be made suitable to interact with a variety of tissue types and occupy physical states ranging from soft hydrogels to stiff scaffolds [9]. Tuning the physical and biochemical properties of materials provides levers of control that limit interactions materials have with host tissues [7, 10]. In addition to serving as a delivery vehicle for localizing particles and controlling cell interaction, material systems shield therapeutic agents from the immune system while providing a favorable microenvironment to increase viability [4, 11]. Furthermore, material scaffolds can potentially serve as an integration platform for the host tissue in larger-scale tissue regeneration. Because of these features, a large number of materials have been used for many different modes of gene delivery, addressing a broad variety of regenerative and disease treatment applications. In summary, this chapter will review strategies of gene therapies used in conjunction with engineered systems in the context of (1) tissue regeneration and (2) targeted disease treatment—focusing on revascularization and neurodegenerative disease treatment, respectively. Revascularization was chosen as a representative focus of tissue regeneration as it is a critical element required in, and preceding, all regenerative medicine strategies due to oxygen, nutrient, and immunological requirements of targeted tissues. In addition, revascularization serves as a marker in preliminarily determining proper function and integration of the engineered system into host tissues. Neurodegenerative disease was chosen as a representative of targeted disease treatment due to the difficulty associated with treating brain-associated diseases by both traditional and more modern, advanced approaches. One of the largest hurdles to treat neurodegenerative disease treatment is the blood-brain barrier (BBB)—a network of tight junctions between capillary endothelial cells in the brain that serves as a means of protecting the brain from toxins and pathogens. Though an integral aspect of the brain’s immune system, this physiology makes treating brain-associated disease difficult, often forcing therapeutics to be applied in large dosages that, when systemically applied, are largely neutralized and cleared by highly metabolic tissues such as the liver.

2  Current prospects of gene therapies As stated above, currently there are five gene therapies approved by the FDA and licensed for use in the United States. Prior to these ­authorizations, there were three approved by the European Marketing



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Authorization (EMA) [2]. All approved vectors so far in both the United States and Europe utilize viral gene therapy vectors [2]. The EMA approved the first gene therapy in November 2012 for use in Europe. Glybera (alipogene tiparvovec) (uniQure; Amsterdam, Netherlands) is a direct gene therapy utilizing an adeno-associated virus serotype 1 (AAV1) vector delivering the gene responsible for the expression of lipoprotein lipase (LPL). Early phase trials of Glybera showed that 50% of the enrolled subjects reached primary efficacy end points in ­comparison to the baseline, and clearance triglyceride content was similar to that of normal subjects—suggesting metabolic efficacy and reduction of pancreatitis over a 3-year period [12]. In addition, no adverse events reported besides a mild immune response to the vector capsid and irritation around the injection site [12]. Despite initial success, long-term follow-up showed loss of effectiveness of the LPL transgene delivered [12]. The EMA approved a second gene therapy, Imlygic (Talimogene laherparepvec), in October 2015. At the same time, Imlygic was also the first ever gene therapy approved by the FDA. Imlygic is a direct gene therapy that utilizes a modified form of the herpes simplex virus type 1 as a localized treatment of unresectable cutaneous, subcutaneous, and nodal lesions for melanoma patients with recurring melanoma postsurgical intervention [13, 14]. Imlygic selectively replicates in tumor cells and produces granulocyte macrophage colony-stimulating factor (GM-CSF), resulting in recruitment and activation of antigen presenting cells leading to subsequent tumor-specific T-cell responses by the host immune system [15]. A 436 patient phase 3 trial of Imlygic showed significantly higher durable response rates to Imlygic versus subcutaneous injection of GM-CSF [15]. Minor adverse events were present in response to Imlygic—including nausea, chills, influenza-like symptoms, and injection site pain [15]. A year after approving Imlygic, the EMA approved Strimvelis (GlaxoSmithKline; Middlesex, United Kingdom); an indirect gene therapy utilizing a retroviral vector to treat severe combined immunodeficiency caused by adenosine deaminase deficiency (ADA-SCID). The first indirect gene therapy approved by the EMA, Strimvelis is a cell therapy that requires the isolation of CD34+ stem cells that are transduced with a retrovirus delivering the human ADA minigene [16]. These cells were then cultured before returning the cells to the subject [16]. There was a reported 100% survival rate over the course of 6.25 years, a decreased number of severe infection rates, and overall increased immune response in ADA-SCID subjects [17]. It’s important to note that the vast majority of the subjects showed neurological responses during treatment or follow-up [17]. In late August 2017, the second gene therapy was approved for use in the United States by the FDA. Kymriah (tisagenlecleucel) (Novartis Pharmaceuticals Corporation; Basel, Switzerland) is an indirect gene therapy used to treat children and young adults with B-cell lymphoblastic

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leukemia. Kymriah uses a replication-incompetent lentivector (LV) to transduce autologous T cells with a gene cassette carrying a chimeric antigen receptor (CAR) with a CD3-zeta domain—thus providing an activation signal and CD137 domain costimulatory signal [18]. Phase 1–2a studies showed persistent remission and relapse-free survival rates of 81% and 80% respectively, without the need for additional infusions [18]. Despite the success of Kymriah cell therapy in causing remission in treated groups, side effects such as neurologic adverse events as seen in other anti-CD19 CAR T-cell therapies were observed but mostly transiently [18–20]. The third FDA gene therapy approval came soon after in October 2017. Yescarta (axicabtagene ciloleucel) (Kite Pharma, Incorporated; Santa Monica, California, United States), is an autologous anti-CD19 CAR T-cell therapy for patients with relapsed or refractory large B-cell lymphoma that has failed to respond to conventional therapy options [21]. Similar to Kymriah, Yescarta is an indirect gene therapy that utilizes an integrating vector system via a replication-incompetent vector—in this case a retrovirus. In a phase 2 study, 82% of the 101 patients treated had an objective response to the treatment, and 54% had a complete response [21]. Though also showing promising results, side effects including myelosuppression, cytokine release syndrome, and neurologic events persist (at 78%, 13%, and 28%, respectively) [21]. The fourth FDA gene therapy approval occurred in December 2017 and was given to Luxturna (voretigene neparvovec-rzyl) (Spark Therapeutics, Incorporated; Philadelphia, Pennsylvania, United States) for the treatment of biallelic RPE65-mutation-associated retinal dystrophy—a degenerative condition that leads to total blindness. Luxturna is the second direct gene therapy ever approved by the FDA and is the first inherited disease gene replacement therapy of its kind, utilizing an adeno-associated virus serotype 2 (AAV2) vector to deliver functional copies of the RPE65 cDNA with a modified Kozak sequence to retinal pigment epithelial cells [22]. Phase 3 clinical trials showed that a majority of the 20 patients receiving Luxturna showed better vision quantified by multilevel mobility tests (MLMT), visual field testing, and full-field light sensitivity threshold (FST) testing, improvements not seen in the control group. In addition, no severe ­product-related adverse events or immune responses were observed [23]. The fifth and most recent gene therapy approval by the FDA came in May 2019 for Zolgensma (AveXis, Chicago, Illinois, USA) for the treatment of pediatric spinal muscle atrophy 1 (SMA1)—a genetic neurodegenerative disorder that leads so paralysis and death of children affected at a rate of 90% by age 2 [23a]. Zolgensma is the third direct gene therapy approved, and the first ever systemically applied direct gene therapy ever approved by the FDA. Zolgensma utilized a derivative of AAV serotype 9 (AAV9) that preferentially transduced neural tissues with high fidelity, and delivers the survival motor neuron 1 (SMN1) gene deficient within



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cells of those affected by SMA1 [23a]. A phase 1 clinical trial of 15 children with SMA1 showed incredible promise for the therapeutic—with sharp increases on the CHOP INTEND scale at 1- and 3-months following gene delivery compared to declines in historical cohorts [23a]. Of the 12 patients who had received the higher dose of Zolgensma, 11 sat unassisted, 9 rolled over, 11 fed orally and could speak, and 2 walked independently—activities SMA1 patients at this stage of disease progression are historically incapable of doing. At 20 months post administration no events were seen, and, most remarkably 100% survival of all 15 patients was maintained in contrast to historical cohorts reporting only 8% survival [23a]. Though all these therapeutics were landmark in that they were the first of their kind to receive regulatory approval, hurdles to their extensive application in the therapeutic marketplace still exist—the largest of which being their costs. Glybera, once celebrated and hailed as a beacon of progress for gene therapy in the Western world, now serves as a grim reminder for those advancing the gene therapy field. Glybera set a record-breaking price nearing $1,000,000 per treatment—a price that caused its demise in the competitive pharmaceutical market in 2017, only 5 years after its initial approval [24]. All other gene therapies currently on the market barring Zolgensma have costs below the seven-figure price Glybera set but are still markedly high. While under evaluation, Imlygic was initially categorized as not cost effective by the National Institute of Clinical Excellence and was forced to develop a patient access scheme with the US Department of Health in order to ensure their treatment maintain minimum competitiveness in its target market [13]. As a result, the current cost for a single dose of Imlygic is the lowest among the approved gene therapies at $65,000 a treatment [25]. Strimvelis has a cost of approximately $670,000 per treatment—however, this is a low price when compared to necessary lifelong treatments for ADA-SCID [13]. Kymriah is marketed as a single treatment, curative option and is priced at $475,000, and Yescarta has a wholesale acquisition cost of $373,000—prices far above the price range of more traditional cancer treatments [26, 27]. Luxturna, which has a relatively small market of potential patients and could therefore categorized as a niche treatment, has a current price of $425,000 per eye [28]. Zolgensma broke Glybera's previous record and became the most expensive therapeutic on the marketplace, with a cost of $2,100,000 per treatment with the possibility of yearly installments of $425,000 being discussed as of 2019 [28a]. What makes these therapeutics competitive is their potential of offering curative treatment options in exchange for a large one-time cost—however, only time will tell whether current value frameworks will allow these novel treatments to survive in the highly competitive and ever-expanding therapeutic marketplace. In addition to the 3 EMA and 5 FDA approved therapies, there are over a hundred gene therapies that have participated in late-stage clinical ­studies worldwide—with 98 gene therapies in phase 3 clinical trials, and

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Publications (n)

Adenovirus

Lentivirus Plasmid Adeno-associated virus

(A)

Vectors used in clincial trials (%)

4.  Bioengineering strategies for gene delivery

Adenovirus Plasmid

Adeno-associated virus Lentivirus

(B)

FIG. 4.2  Recent scientific publication and clinical trial trends for common gene therapy vectors. While publication levels of plasmid gene therapies have remained relatively stable and adenovectors have dropped, both adeno-associated vectors and lentivectors have demonstrated increased publication levels in recent years, as determined from the PubMed database (A). Similar trends are seen in gene therapy clinical trial numbers for these vectors as compiled from a series of reviews [2, 29, 30] (B).

3 in phase 4 clinical trials as of 2017—utilizing vectors ranging from simple plasmids to complex integrating viral vectors [2] (Fig. 4.2). Currently, there are six gene therapies in active stage 3 clinical trials taking place that have been reported to the NIH [31].

3  Vector design A broad variety of vectors is available for use in regenerative and disease treatment applications. Each vector has inherent strengths and weaknesses, and in order to make a selection of what vector best suits the need, several parameters must be considered—including the cell target, desired expression duration, immune tolerance, delivery efficacy, and biosafety. Both viral and nonviral vectors have been utilized in gene therapy applications, some of the most common of which are summarized in Table 4.1 and described below. Viral vectors have a variety of sizes, morphology, surface biochemistries, and therefore exhibit unique biotransport characteristics in both tissue environments and in solution [7]. Viral vectors also have high levels of gene transduction efficacy. However, biosafety concerns persist regarding their use due to the potential of some to cause oncogenesis or a severe immune response [37, 53]. Nonviral vectors, in contrast, exhibit lower safety concerns, but also possess lower efficacy in successful transgene introduction.

3.1  Viral gene therapy Many viruses have been studied for gene therapy applications, however, the most common include retroviral and LVs, adenovectors, and ­adeno-associated vectors—a detailed account of which is covered below in more detail.



TABLE 4.1  Common gene therapy vector information and features Diameter (nm)

Gene size limit (kb)

Particle surface

Gene presence

First gene therapy publication (year)

Total clinical trialsa

Retrovirus and LV

80–120 [32, 33]

8 [7]

Envelope [34]

Integrative [35]

1984 [36]

674 [2]

Ad

70–100 [37–39]

8 [40]

Capsid with fiber projections [7]

Episomal [37]

1990 [41]

547 [2]

AAV

20–30 [42–44]

5 [45]

Capsid [46]

Episomal [42]

1984 [47]

204 [2]

Plasmid

Radius of gyration (nm) 2.7–6 kb Linear 115–235 Supercoil 86–146 [48]

>10 [49, 50]

–

Episomal [51]

1980 [52]

442 [2]

3  Vector design

Vector

a

Represents total number of clinical trials worldwide as of 2017.

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3.1.1  Retroviral and lentivectors Retroviral vectors are genome integrating vectors that, after integrating into the host’s DNA, enter the cell lifecycle and replicate along with the host genome. LVs are a genus of the Retroviridae viral family that along with being able to transduce not only dividing cells, as many retroviruses can but also nondividing cells as well [54]. Specifically, LVs are derived from the human immunodeficiency virus (HIV), which utilizes an enveloped capsid to introduce its diploid RNA genome to the host cell. LV have a heterogeneous diameter typically ranging from 80 to 120 nm, possess a diploid RNA genome of approximately 9 kb, and can be used to deliver genes as large as 8 kb [7, 32–34]. In order to increase the biosafety of these vectors for use in therapeutic applications, several modifications to the wild type virus have been made. For example, to produce LV for gene therapy the vector genome is split into multiple plasmids that are transfected into a producer cell line—typically human embryonic kidney 293 T cells—where viral particles are assembled [55–57]. Second- and third-­ generation LVs exist utilizing three and four plasmids, respectively, to produce LV with strong safety profiles [55]. All plasmids used to create LV particles contain partitioned segments of the LV genome essential for replication or packaging information—but only a single plasmid contains a packaging signal, leading to the production of transduction competent LV viron loaded solely with the plasmid carrying the gene of interest [55–57]. Key viral replication genes, gag, pol, tat, and rev genes are absent in the final LV particles, thus the vectors produced are replication deficient [7, 55–57]. Infectivity spectrums and biosafety profiles are further strengthened by (1) the elimination of promoter/enhancer sequences in the 3′ long terminal repeats thus creating self-inactivating (SIN) vectors, and (2) the use of a nonnative viral envelope—the most common of which being the vesicular stomatitis virus glycoprotein (VSV-G) [7, 53, 58]. As of 2017, 196 clinical trials utilized LVs [2]. LVs are desirable for gene therapy applications for several reasons. LVs are capable of transducing both dividing and quiescent cells, can carry a gene expression cassette of 8 kb, and low immunogenicity have been reported [59–61]. Furthermore, due to the vector’s integration ability, long-term transgene expression is possible [61]. In addition to being the first ever vector construct utilized as an approved gene therapy by FDA, LVs have been the focus of many preclinical protocols in applications to treat genetic diseases, cancers, and vascular diseases [7]. LV also possesses features that lend caution to their use. Integration of the viral gene cassette has the potential of activating oncogenes, disrupting active gene expression, silencing gene expression, and modifying histones [53, 62–64]. In addition, histone modification can lead to the inactivation of the LV transgene—inhibiting long-term therapeutic gene expression and hindering potential therapeutic gain [65]. These challenges



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have limited the potential applications of LV but also motivated the engineering interventions described more in detail in the following sections of this chapter. 3.1.2 Adenovectors Adenovirus (Ad) is a nonenveloped, 36 kb, double-stranded DNA virus of the Adenoviridae family that has a 70–100 nm diameter icosahedral protein capsid encapsulating a nucleoprotein core [7, 37, 66, 67]. Seven subgroups and 57 serotypes of Ad have been identified, the most used in gene therapy being Ad5 [7]. Ad vectors are able to transduce transgenes as large as approximately 8 kb in both dividing and nondividing cell types and possess a strong, but transient, gene expression profile [7, 40, 68]. As of 2017, Ad was the focus of over 20% of total gene therapy clinical trials, representing a total of 547 trials [2]. Adenovectors are very effective viral vectors for gene transfer but they also have several features that caution their use in gene therapy applications. First, Ad transduction is dependent on CAR receptor interaction, which leads to a preferential transduction in liver tissue [7, 69]. Second, Ad biological activity diminishes over time in vivo, presenting a limited time for transgene expression [7, 70]. Finally, Ad capsid proteins are highly immunogenic, causing severe immune responses in some subjects [7, 69]. Notably, in 1999, an adenovector used in a phase 1 clinical trial at the University of Pennsylvania led to the first death directly associated with a gene therapy due to a severe immune response in the subject treated [71]. Because these features that potentially limit Ad use were seen early on, Ad therapies were one of the first to employ the use of engineering strategies to address and curb the effects of these shortcomings [72]. Continuing efforts to increase localization and decrease immunogenicity through material strategies is ongoing and use strategies ranging from encapsulation to surface coatings [7]. 3.1.3  Adeno-associated vectors Adeno-associated viruses (AAV) are members of the Parvoviridae family that require Ad virus coinfection for proper replication [73–75]. AAV possess a 4.7 kb single-stranded DNA genome inside a 20–30 nm diameter icosahedral protein capsid [42–44, 74]. The AAV genome has rep and cap reading frames located between inverted terminal repeats (ITRs) that together are responsible for viral nuclear localization, integration, transcription, assembly, and replication [76–81]. For use in gene therapy applications, rep and cap are replaced by the target gene, which can be as large as 5 kb [45]. Though integration is possible, it occurs in low frequencies and delivered genes exist predominantly in an episomal manner [42]. In addition, AAV capsids can be interchanged to create unique artificial hybrid AAV, and research continues in expanding and diversifying p ­ rotein

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capsids through directed evolution for better and more refined tissue targeting capabilities [82]. There are 204 clinical trials utilizing AAVs in progress as of 2017, and a burgeoning interest in AAV gene therapies is growing several reasons [2]. AAVs present a long list of advantages, but some challenges exist for their usage in clinical applications. First, some of the advantages are listed here. Foremost, AAV, unlike the LV and Ad viruses previously mentioned in this chapter, are not pathogenic [83–86]. Second, long-term expression is possible in nondividing somatic cells due to a stable episomal conformation [42]. Third, targeting certain tissues is possible due to 12 naturally occurring serotypes and hybrids that favorably transduce-­specific cell types [82, 87]. Despite these benefits, AAV also denotes several challenges. Upon exposure to AAV therapies, antibodies against them form—­ effectively neutralizing administered AAV therapies and compromising their efficiency if prolonged AAV gene therapy is necessary [88]. AAV gene therapies may also be neutralized upon initial administration by antibodies formed by exposure to AAV the patient encountered in their everyday lives [88]. This uncertainty continues to motivate innovation in AAV therapies and their delivery and necessitates the employment of bioengineering strategies. Consequently, many naturally occurring and synthetic biomaterial carriers have been utilized in AAV delivery—including nanoparticles, scaffolds, and hydrogels [7, 89].

3.2  Nonviral gene therapy Circular double-stranded DNA called plasmids (pDNA) replicate independently of chromosomal DNA. pDNA can be produced to contain transgenes exceeding 14 kb and can exist in linear or supercoiled conformations [50]. pDNA size can best be estimated by their radius of gyration and depends on several factors such as plasmid conformation and amount of base pairs—with a 6 kb plasmid having a radius of gyration between 86 and 235 nm [48]. There are many features that make pDNA vectors desirable: (1) they are able to contain long and complex genetic elements, can carry multiple genes; (2) have long-term storage capabilities; (3) have low toxicity; (4) have a strong safety profile; and (5) are easily produced at a comparatively low cost [7, 50, 90, 91]. Due to these features and the large-scale production capabilities of these vectors, they have been largely studied for gene therapy applications—with 442 clinical trials taking place as of 2017 [2]. There are several limitations that persist with pDNA vectors used in gene therapy. pDNA is amplified by Escherichia coli bacterial strains designed to minimize DNA degradation and recombination. While bacterial production vehicles are easy to maintain, genetic elements essential for replication of the plasmid bacterial cells can cause immune responses in



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mammals [90]. pDNA has a low transfection efficiency due to their negative charge that electrostatically repels them from cell membranes, and, in addition, pDNA vectors also lack the cell targeting ability that viral vectors have [7, 90]. Transgene expression in targeted tissues, therefore, is low. Furthermore, for cells that are successfully transfected, transgene expression is fleeting and tends to decline rapidly to negligible levels in a period of days [7]. Advancements have been made in order to increase transfection and gene expression profiles that range from redesigning the plasmid itself to implementing physical techniques to better therapeutic outcomes. pDNA designs to increase efficiency and transgene expressions including nonviral integrating vectors (i.e., Sleeping Beauty Transposons) and minicircles have been developed [92–94]. Physical intervention methods have also become popular and involve creating cellular pores in targeted tissues—sonoporation and electroporation are two common methods, using ultrasound and electric fields, respectively [7]. Biomaterial delivery strategies including nanoparticles, hydrogels, and scaffolds have also been used to localize pDNA and pDNA transfected cell delivery [7, 95–98].

4  Delivery strategies In order to address inherent vector limitations, a strategy that involves the use of a delivery system could be envisioned that takes into consideration not only the vector of interest but also the tissue target and mode of gene delivery (direct or indirect). Delivery systems allow for vector localization—concentrating gene therapy vectors to the region of interest [7]. In addition to localizing the vectors in targeted tissues, delivery systems also serve to shield the vectors from deactivation and destruction by the immune system [99]. In protecting gene therapy vectors from immune action, transduction or transfection efficacy is increased, dosage is better defined, and lower levels of the vector can be used for the desired effect. Numerous potential delivery strategies exist that encompass a spectrum of physical characteristics, allowing for precise control over the material selection that suits the need presented by the vector, tissue, and desired release kinetics. Delivery strategies can be designed for broad applications encompassing advanced tissue engineering approaches to simple drug delivery [99–103]. Biomaterials provide mechanical structure, a means of controlling cell signaling, and can be designed to support cells in the formation of tissues [102, 103]. Tissues and their extracellular matrices (ECM) vary in structure and physical characteristics that allow for proper cell communication and function. This variability motivates the design of biomaterials that can effectively mimic local ECM and thus support tissue growth in regenerative applications [103]. Drug delivery applications

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of materials focus on the encapsulation and controlled release of loaded particles [101]. Designing the appropriate system must take the physical properties of both the loaded particles and material into account to modulate mass transport effects [7]. Several parameters influence the encapsulation efficiency and release rate, including the size of the loaded particles, the intrinsic chemistry, the material degradation rate, and the mesh size of delivery vehicle [102]. Additional strategies of gene delivery, including the utilization of particle coatings, nanoparticles, lipoplexes, polyplexes, and lipid-polymer complexes, are also important to consider but they fall outside the scope of this chapter, despite being extensively studied and reviewed elsewhere [104–110]. These considerations must all be taken into account for gene therapy, and vary depending on the delivery method. Direct gene therapies deliver vectors in vivo, therefore delivery strategies for this method encapsulate vectors for the purpose of either delivering vectors to tissues near to the material carrier, or supporting cell infiltration into the material where gene delivery subsequently takes place [35, 111]. Each vector class presents unique biochemistries that favor interactions with specific proteins, proteoglycans, and sugars, and these affinities determine the cells vectors are able to interact with, therefore impacting transduction. Therefore, delivery strategies must take these biochemistries into account to predict vector/material interactions and promote vector retention [112, 113]. Vectors have also been successfully chemically modified for increased control of vector release [112, 113]. Safe and efficient dosing in delivery strategies requiring the release of viral vectors to adjacent tissues necessitates precise spatiotemporal presentation of vectors [7]. Indirect gene therapies involve gene transfer outside the body and therefore manipulation of cells ex vivo is present prior to subsequent application of cells to the area of interest. For bioengineering approaches to the delivery of indirect gene therapy, materials that support cell interactions, such as adhesion, motility, and infiltration are necessary. Cellular interaction with materials requires interactions between receptors on cell surfaces and polymers exhibiting specific amino acid sequences that recapitulate native extracellular matrix motifs either innately or through chemical modification of the material [4, 7, 103, 114]. Specifically, the amino acid sequence ­arginine-glycine-aspartic acid (RGD) represents one of the most commonly used peptides for promoting cell adhesion in biomaterial systems [4, 103, 114]. Both naturally occurring and synthetic biomaterials have been successfully employed in gene delivery applications.

4.1  Synthetic materials Synthetic polymeric materials are obtained via chemical synthesis and have the advantage of being highly reproducible [4, 7]. In addition,



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production of these materials can be precisely manipulated to obtain a platform with highly tuned physical and biochemical characteristics such as mechanical strength, cell recognition response, and degradation kinetics [7]. Synthetic materials can also be formulated with the intention of not being highly immunogenic [4]. A large variety of synthetic materials have been applied for biomedical purposes including gene delivery, however, two of the most common, poly(ethylene glycol) (PEG) and ­poly(lactic-co-glycolic) acid (PLGA) will be covered here. 4.1.1  Poly(ethylene glycol) PEG is one of the most commonly used polymers for bioengineering applications and has been utilized in medical devices, drug delivery devices, and tissue engineering scaffolds [4, 115]. PEG is biologically inert, hydrophilic, biocompatible, tunable, and easily chemically altered—making it a desirable material for biomedical uses [4, 116]. PEG can be predictably synthesized in a variety of molecular weights, allowing for precise control over physical and mechanical properties [4]. PEG cross-links are broken down in by hydrolysis in vivo, and these hydrolyzed PEG chains are able to be successfully excreted by the body through the renal system—as a result PEG is categorized as a biodegradable material [4, 115]. PEG is also able to be blended with other polymeric substances to create functional and tunable composite systems with unique degradation kinetics [4, 117]. Because of these desirable features, PEG has been extensively utilized as a delivery vehicle for therapeutic cargos, including viral and nonviral genetic vectors [118–121]. For indirect gene therapy applications, PEG requires modification to successfully interact with cells in delivery and tissue engineering applications [4, 122, 123]. PEG has been successfully synthesized and chemically modified with cell adhesive and protease substrate peptide sequences to create ECM mimics that support cell infiltration, furthering applications of PEG in tissue engineering [4, 122, 123]. In addition to being utilized as a delivery vehicle, PEG has also been extensively utilized as a coating substance for gene therapy vectors, shielding them from serum components that can stimulate clearance or immunogenic action [7, 34, 112, 124–128]. 4.1.2  Poly(lactic-co-glycolic) acid PLGA is another frequently used material for applications in drug delivery and tissue engineering [4, 129]. Similar to PEG, PLGA is biocompatible, biodegradable, hydrophilic, and has tunable physical and mechanical properties for biomaterial applications in a variety of tissues. Tunability of PLGA is largely due to the copolymer nature of the substance—its physical and mechanical properties can be varied by altering the ratio of polylactic acid (PLA) to polyglycolic acid (PLG) [4, 130]. PLGA has ester linkages that are points of degradation via hydrolysis,

122

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and degradation products can then be easily removed from the body [4, 129, 131]. Because of these properties, PLGA is a desirable delivery vehicle and has been used in direct gene therapies delivering viral and nonviral vectors [132–134]. Though PLGA is an FDA approved drug delivery strategy for parenteral administration, PLGA is not supportive of cell adhesion and is not easily chemically modified—therefore, tissue engineering applications involving PLGA scaffolds have relied on immobilizing biomolecules to PLGA by covalent techniques to establish cell adhesion [4, 129, 135, 136]. The ability of PLGA to have tunable characteristics including cell interactions has led to its use in indirect gene therapies for cell delivery and infiltration [137–139].

4.2  Naturally occurring materials Naturally occurring polymers, in contrast to synthetic polymers, are those isolated from the plant, animal, or human sources. Naturally occurring polymers, due to their natural derivation, have structures and biochemistries similar to that of native ECM [7]. However, also due to natural sourcing, batch variability persists and reproducibility of structures made from these materials represent a challenge and it is something to consider [4]. In addition, certain naturally occurring materials can contain molecules that may elicit an immune response when applied in  vivo. Many naturally occurring polymeric materials have been applied for gene delivery applications and will be mentioned in this chapter, but alginate, fibrin, collagen, and gelatin, will be specifically described below. 4.2.1 Alginate Alginate is a negatively charged block copolymer that can be isolated from either bacteria or brown algae and it is composed of a-l-guluronic (G-block) and B-d-mannuronic (M-block) acid residues [4]. Current commercially available alginates are isolated from algae and typically have an average molecular weight of approximately 250 kDa [4, 140]. M-block and G-block composition determines the physical and chemical properties of the alginate copolymer, and due to its sourcing diversity and variations in extraction methods, batch variability can persist [4]. Alginate is highly biocompatible and has tunable physical properties that make it desirable for many biomedical applications, including gene and drug delivery [35, 141, 142]. Alginate has a gentle gelation procedure, and as a result, is able to be applied in a minimally invasive manner. Direct application of viral vectors via injectable alginate hydrogels has shown to localize transgene expression [35]. Other gene therapy applications of alginate hydrogels have been utilized viral and nonviral vectors [143–145]. Alginate is biologically inert and, without modification, does not support cell adhesion. Chemical modifications via the addition of peptides have resulted in cell



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supportive alginates [146–148]. In addition, alginate is also nondegradable. Degradable alginates can be made via chemical modifications to make the polymer susceptible to hydrolysis (i.e., oxidation via sodium periodate) or by using enzymes active against alginate (i.e., alginate lyase) [149, 150]. 4.2.2 Fibrin Fibrin is a globular protein derived from humans and animals that forms the basis of the blood clotting process [151]. Produced largely by liver tissue, fibrin, and its precursor molecule fibrinogen have both been studied and applied extensively in biomedical applications due to its simple isolation and ECM similarity [4, 152]. Fibrinogen is a large protein of approximately 350 kDa consisting of two identical subunits, each consisting of three polypeptide chains [151]. Many commercial forms of fibrinogen exist isolated from animal sources including murine, bovine, and primate sources—and purified human fibrinogen is a sealant approved for use by the FDA. In vivo, fibrin is a protein that forms spontaneously when fibrinogen is cleaved by thrombin when tissue injury occurs and is degraded by plasmin when tissue repair takes place [4, 153, 154]. Physical and chemical properties of fibrin polymer products can be tuned by controlling pH, ionic strength, or concentration of the fibrinogen precursor during the polymerization process [4, 155]. Due to fibrin’s animal and human sourcing, scaffolds are biocompatible, resorbable, and naturally cell adhesive—supporting cell infiltration of a variety of cell types including those that are involved in forming natural ECM [4, 156]. Because of these features, fibrin has been used extensively in deliverables, clinical tissue engineering applications, and both direct and indirect gene therapy applications [4, 95, 157–159]. 4.2.3  Collagen and gelatin Collagen is the chief structural protein in animal tissues and accounts of 30% of vertebrate body protein [160]. Collagen is a broad family of proteins and though 13 types of collagen have been identified, collagen type I, mainly found in skin, tendon, and bone, is the most commonly used in biomedical applications [160]. Gelatin is a polymer sourced from collagen type I that has been denatured via acid treatment or alkaline hydrolysis [4, 160]. Common commercial sources include porcine and bovine tissues, and due to the processing and source variation, gelatin molecules vary broadly in size. While seen most extensively in food products and consumables, gelatin also has generally been recognized as safe (GRAS) by the FDA, has excellent biocompatibility, and is easily chemically modified due to the prevalence of a variety of functional groups [4, 161, 162]. In addition, adhesion peptides such as RGD are naturally present in collagen and gelatin, and therefore cell adhesion is supported [162]. Gelatin has thermosensitive and has thermo-reversible cross-links that are broken at physiological temperatures, therefore for in vivo biomedical applications

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additional cross-linkers should be introduced in order to maintain the physical integrity of the polymeric structure [163]. Because of the biosafety and modification ability of collagen and gelatin materials, they have been utilized in direct and indirect gene therapies for both simple delivery and tissue engineering applications [164–168].

5  Gene delivery via biomaterial strategies 5.1  Revascularization Cardiovascular diseases have remained the leading cause of morbidity and mortality worldwide for more than a century [4, 169–171]. Vascularization is essential for providing and maintenance of tissue homeostasis and loss of vascularization can lead to not only loss of tissue function but also tissue necrosis and death. Many physiological dysfunctions arise from or are exacerbated by lack of proper vascularization, including peripheral artery disease (PAD), atherosclerosis, arthritis, and diabetes [4, 171, 172]. Current therapeutic options and intervention strategies are not curative but simply aimed to improve quality of life and modulate disease progression [4, 170]. Standard care options first aim to reduce risk with lifestyle changes while presenting pharmacological treatment strategies before intervening surgically with either bypass or endovascular revascularization [4, 170, 173]. However, for those patients who fail to respond to these first-line therapies, alternatives are sparse and the rate of success is low, resulting in high mortality rates [174]. These challenges have motivated the development of alternative therapeutic strategies that drive new vascularization is ischemic regions [4, 175, 176]. Therapeutic angiogenesis utilizes angiogenic factors (including VEGF—vascular endothelial growth factor) or cells to promote the formation of new blood vessels to revascularize ischemic tissues and is a promising developing field for cardiovascular disease treatment [4]. Polymeric materials have been commonly used as delivery vehicles for the localized controlled release of therapeutics and/or integration platform for host tissues in tissue engineering. Biomaterial scaffolds have extensively been used as a means of controlling the delivery of proteins, genes, and cells in revascularization efforts [132, 141, 142, 144, 177, 178]. Loaded scaffolds are applied to implantation sites where they, and the therapeutics loaded into them, are allowed to interact with host tissues [179–181]. In order to establish a successful vascular network in or around tissue constructs, numerous humoral and cellular signaling mechanisms must take place in the proximity of the host tissue and implant interface [179]. Many different therapy vectors combined with material strategies have been applied toward revascularization purposes, a summary of which are shown in Table 4.2 and are organized by vector below.



TABLE 4.2  Revascularization strategies utilizing biomaterial carriers and gene therapies Gene therapy

Biomaterial/tissue integration

References

Alginate

Hydrogel

Direct

No

[143]

Collagen

Hydrogel

Indirect

Yes

[165]

Gelatin

Scaffold

Indirect

Yes

[164]

PEG

Hydrogel

Direct

No, Yes

[118, 119]

PLGA

Scaffold

Direct

Yes

[133]

PF

Hydrogel

Indirect

No

[182]

β-TCP

Scaffold

Indirect

No

[183]

Alginate

Microcapsule

Indirect

Yes

[145]

Collagen

Hydrogel

Indirect

Yes

[166]

Fibroin

Film

Indirect

No

[184]

Synthetic

PLGA

Scaffold

Indirect

No, Yes

[137–139]

Natural

–

–

–

–

Synthetic

PEG

Hydrogel

Direct

No

Biomaterial

Retrovirus and LV

Natural

Synthetic

Ad

AAV

Natural

5  Gene delivery via biomaterial strategies

Physical state

Vector

[185] Continued

125

126

TABLE 4.2  Revascularization strategies utilizing biomaterial carriers and gene therapies—cont’d Biomaterial

Plasmid

Natural

Synthetic

Physical state

Gene therapy

Biomaterial/tissue integration

References

Alginate

Hydrogel

Direct

No

[144]

Collagen

Scaffold

Direct

No

[168]

Microspheres

Direct

No

[186]

Sponge

Indirect

Yes

[187]

Elastin

Hydrogel

Direct

No

[188]

Fibrin

Hydrogel

Direct

No

[158, 159]

Matrix

Indirect

No

[95]

Gelatin

Hydrogel

Direct

No

[167]

HA

Hydrogel

Direct

Yes

[189]

PLGA

Scaffold

Direct

Yes

[134]

Sponge

Direct

No

[132]

4.  Bioengineering strategies for gene delivery

Vector



5  Gene delivery via biomaterial strategies

127

5.1.1 Lentivectors Direct delivery of LV carrying proangiogenic factors from alginate material platforms has shown promise in multiple in vivo models. For example, in chick chorioallantoic membrane (CAM) assays, CAMs exposed to alginate microgels loaded with LV encoding the VEGF gene showed a statistically significant increase in vascularization when compared to LV bolus controls and showed a similar response in VEGF protein bolus [143]. In murine models, direct delivery of VEGF coding LV from synthetic hydrogels showed mixed results. For instant, subcutaneous PEG implants delivering LV VEGF had no significant increase in vascularization in the implanted region compared to empty gel controls [118]. In contrast, it has been demonstrated that PEG hydrogels loaded with LV encoding VEGF did significantly increase revascularization with vascular infiltration of the porous implants implanted subcutaneously in mice [119]. Furthermore, other biomolecules delivered by LV outside of VEGF have also shown increased angiogenic response potential. PEG hydrogels delivering LV encoding sonic hedgehog (Shh) did significantly increase vascularization compared to empty controls without tissue integration when applied subcutaneously [118]. PLGA scaffolds delivering LV encoding platelet-derived growth factor-BB (PDGF-BB) also significantly increased angiogenesis and tissue integration in murine bone cranial defect models [133]. Indirect methods of LV gene delivery used in conjunction with both naturally occurring and synthetic biomaterials have also shown increased revascularization if multiple tissue types. Bone mesenchymal stem cells transduced with LV encoding VEGF upregulation factor hypoxia-­ inducible factor-1α (HIF-1α) showed enhanced local neovascularization as compared with blank controls and when loaded into gelatin sponge scaffolds and implanted into calvarium defects of mice [164]. Likewise, subcutaneously applied collagen hydrogels loaded with genetically modified adipose-derived mesenchymal stem cells (adMSCs) carrying LV delivered angiogenic extracellular matrix protein developmental endothelial locus-1 (DEL-1) showed significantly increased tissue integration and vascularization when compared to groups modified only with a reporter gene [165]. Revascularization is also seen in indirect LV gene therapies applied with blended polymer platforms, such as PEG-fibrinogen (PF) copolymers [182]. For example, a PEG vehicle coupled with RGD motifs has been successfully tested and validated for strategies with localizing the cell therapy [182]. Induced pluripotent stem cells (iPSCs) were transduced with LVs encoding placental growth factor (PlGF) and matrix metalloproteinase 9 (MMP9), and upon application to mice displaying myocardial infarction via injectable PF scaffold, showed successful iPSC integration and revascularization of heart tissue [182]. In addition to delivering cells carrying genes encoding for proteins that act to increase tissue ­revascularization,

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indirect gene therapies of LV have also been used to deliver noncoding nucleic acid segments, such as microRNA (miRNA), that have shown increased levels of tissue formation in murine models. Though revascularization was not specifically measured, bone marrow mesenchymal stem cells transduced with LV encoding bone and angiogenesis promoting miRNA miR-26a were seeded into β-tricalcium phosphate scaffolds and implanted into mouse skull defects and showed significant increases in bone volume and suggests increased vascularization is a possible therapeutic application of this vector construct [183, 190]. 5.1.2  Adenovectors and adeno-associated vectors Indirect Ad gene therapies are utilized with biomaterials frequently in revascularization efforts—delivering genetically modified cells in order to establish new blood vessels. Again, both naturally occurring and synthetic polymers have been used in conjunction with Ad modified cells and these strategies have shown promise in revascularizing tissues. For example, collagen gel loaded with urine-derived stem cells (USCs) transduced with Ad-VEGF promoted angiogenesis and infiltration of blood vessels into the subcutaneous implants in a murine model [166]. In addition to significantly improving ischemic tissue function, myogenic markers in the delivered USCs and innervation also were significantly improved when compared to nonmodified controls—suggesting more diverse tissues and better integration of delivered cells [166]. Moreover, organic matrices have also been used to deliver ex vivo gene therapies. For example, one strategy involves using an acellular dermis matrix (ADM) which is a 3D section of decellularized skin. Postcell removal, the remaining scaffold is an intact ECM that can be utilized as a delivery vehicle for biomolecules and or cells for tissue engineering. Ad has been applied to transduce NIH3T3 cells with VEGF, and the resulting cells were encapsulated in alginate microcapsules before being seeded into ADM and placed onto dermal defects of guinea pigs [145]. Cell survival and angiogenesis were significantly different between encapsulated Ad-VEGF groups when nonencapsulated groups, illustrating that the microencapsulation protected the transduced cells and allowed for longer-term proangiogenic biomolecule production and response [145]. Synthetic scaffolds loaded with cell lines producing VEGF have also shown promise in vascular tissue regeneration. PLGA scaffolds loaded with adipose-derived stem cells (ADSCs) transduced with Ad-VEGF and then implanted subcutaneously into mice showed significant revascularization and infiltration of host cells into the PLGA scaffold when compared to controls, with the most significant angiogenic response being present in groups containing transduced ADSCs and endothelial cells [137]. Likewise, Ad-VEGF transduced MSCs loaded into PLGA scaffolds promoted statistically significant neovascularization significant bone regeneration in the distal radial diaphysis defect rabbit



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model [138]. PLGA scaffolds containing bovine meniscal cells transduced with Ad encoding hepatocyte growth factor (HGF) promoted vascularization of meniscal tissue engineering constructs applied to mice subcutaneously—showing significantly more blood vessels in neocartilage-like tissues than controls [139]. Spider silk-derived fibroin films loaded with fibroblasts expressing Ad delivered angiopoietin-1 (Ang-1) also demonstrated increased vascularization response in both CAM and rabbit corneal models, though not significantly [184]. In contrast to Ad, few biomaterial strategies have been employed for the delivery of AAV for revascularization strategies. StarPEG-heparin hydrogels were soaked in AAVs encoding for stromal cell-derived factor 1α (SDF-1α) before being combined with endothelial progenitor cells (EPCs) for direct gene delivery. Groups exposed to AAV-SDF-1α loaded hydrogels showed significantly increased EPC migration activity in vitro [185]. 5.1.3  Nonviral vectors Biomaterials directly delivering proangiogenic factor encoding pDNA show mixed results in revascularization efforts—though they have been utilized in multiple in vivo models with both naturally occurring and synthetic biomaterial carriers. The pDNA of PDGF B-chain showed increased angiogenic response in a subcutaneous rat model in PLGA sponge groups, but not in bolus controls [132]. Another factor largely studied has been endothelial nitric oxide synthase (eNOS)—a wound-healing factor that has shown to promote endothelial cell migration and has also proven to be essential for angiogenesis due to its upregulation of proangiogenic factors [191]. Naturally occurring hydrogels delivering pDNA encoding eNOS have increased vascularization in subcutaneous mouse models. Browne et  al. used a collagen scaffold delivering eNOS pDNA, and showed statistically significant increases in length and density of blood vessels when compared to nonloaded controls [186]. An elastin-based delivery platform delivering eNOS pDNA was applied in subcutaneous mouse models and mouse hindlimb ischemia models—ischemia models showed functional recovery in the eNOS pDNA elastin hydrogel-treated groups while negative control saline groups showed minimal functional recovery [188]. Gelatin loaded fibroblast growth factor 4 (FGF4)—a proangiogenic factor—pDNA has shown to increase vascular responsiveness when compared to both bolus pDNA controls and empty gelatin control [167]. Fibrin encapsulating nanocondensates made of PLL-g-PEG pDNA encoding HIF1α has shown to initiate proangiogenic responses in cutaneous wounds in both healthy and diabetic mouse models, though not significantly [159]. Direct gene therapies utilizing biomaterial carriers for revascularization focus largely on VEGF pDNA, and have returned mixed results. VEGF pDNA delivered by collagen scaffolds induced an increased angiogenesis response when applied in a subcutaneous mouse models, but did not

130

4.  Bioengineering strategies for gene delivery

show a ­significant difference to empty scaffold and bolus VEGF pDNA controls [168]. Similarly, porous hyaluronic acid scaffolds delivering VEGF pDNA did not show significant increases in blood vessel number per area in a subcutaneous mouse model when compared with a pDNA carrying a reporter gene alone, rather, there was a dependence on pore size on cell infiltration and subsequent transfection [144]. In contrast, PLGA scaffolds loaded with VEGF pDNA have shown to significantly increase cellular infiltration and blood vessel density compared to controls [134]. Indirect applications of nonviral gene therapies have also been investigated, and have shown promising results. Zhou et al. utilized, for example, VEGF and SDF-1 pDNA genetically modified myoblasts seeded in collagen sponges and applied in vivo [187]. When these collagen sponges were applied into back muscle defects in mice, pDNA loaded scaffold groups showed increased capillary bed density, with the highest capillary density arising from the combined transfection VEGF and SDF-1 cell groups [187]. Similarly, implantation of fibrin scaffolds loaded with VEGF transfected preadipocytes showed significant increases in maximal vessel ingrowth distance from implant surface, neovascularization numbers, and vessel area when compared to plasmid negative controls in CAM models [95].

5.2  Neurodegenerative disease Neurodegenerative disease is a broad spectrum of conditions that encompass pathological states that involve the spread of misfolded proteins, neuronal/synaptic loss, and gliosis—which is a universal reactive response of glial cells upon injury to the central nervous system (CNS) [192– 194]. Neurodegenerative disease can be age related, such as Alzheimer’s and Huntington’s disease, or age independent, such as amyotrophic lateral sclerosis (ALS), Parkinson’s disease, and some forms of epilepsy [194, 195]. Neurodegenerative diseases are progressive, and symptoms increase in severity over time as abnormal proteins aggregate in brain tissues— resulting in neurotoxicity [194, 196, 197]. Therapeutic strategies include immune-mediated clearance of pathological protein aggregates, blocking intercellular trafficking, and proteolytic processing of these proteins, inhibiting protein aggregation, and upregulating protective proteins [194, 196, 198]. Traditional treatment options such as oral intake or vascular infusions of therapeutic proteins such as growth factors show low effectivity due to physiological structures, such as the BBB, that limit access to the affected region [199]. The BBB is a network of tight junctions that exist between endothelial cells that make up the capillaries of the brain that shields the brain from toxins and pathological particles [199]. The BBB is highly effective at excluding therapeutics, excluding 98% of low molecular weight drugs and nearly 100% of higher molecular weight drugs [199, 200]. As a result, the BBB is one of the most challenging hurdles potential



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131

therapeutic cargos must successfully infiltrate in order to reach targeted brain tissues [196]. Advanced approaches such as gene therapies have been developed in efforts to treat neurodegenerative diseases that can surpass the BBB. For example, viruses encoding pathogenic gene silencing siRNA have been directly injected into the target brain and spinal cord regions with promising results [201–204]. Pseudotyping of viral vectors with nonnative surface proteins that favor neural tissue interaction, such as the rabies virus glycoprotein, have also been created [205]. This pseudotype favors CNS cell transduction, therefore these viral vectors can be injected intramuscularly and then transported in retrograde to the CNS where they can transduce neural tissues [205]. This method has been applied to treat neurodegenerative diseases and resulted in prolonged, localized exposure to therapeutic biomolecules that increased long-term survival when compared to controls [206, 207]. Material strategies for neurodegenerative diseases have been utilized as localized delivery vehicles for therapeutic proteins, as support scaffolds for cell therapies, and nanoparticle biomolecule carriers to help surpass the BBB [199, 208–210]. Drug delivery devices designed for sustained localized delivery of therapeutic proteins surpass the BBB, but are invasive due to their required implantation and have the problem of depleting a limited source of loaded therapeutic [199]. Many support scaffolds for indirect gene therapies involve cell immobilization and encapsulation within a cavity surrounded by an impermeable membrane [199]. The biomaterial carrier is locally implanted, and the cells encapsulated inside secrete therapeutically active agents in the targeted region [199]. This method, while surpassing of the BBB, is also highly invasive. As a result, new methods of targeted delivery utilizing biomaterials have been developed to assist in therapeutic biomolecule delivery across BBB without invasive surgical intervention. For example, biomaterial nanocarriers assist in spatiotemporal control of therapeutic biomolecule presentation in targeted neural tissues, and these polymeric nanocarriers can be coated in biomolecules that assist in the nanoparticle BBB permeability and increase pharmacokinetic efficiency [199, 211]. However, as previously stated, nanoparticle material carriers are outside of the scope of this chapter. Biomaterial carriers utilized in conjunction with gene therapies have been developed for and shown to beneficially impact several neurodegenerative diseases, as summarized in Table  4.3 and detailed below. 5.2.1 Lentivectors Glial cell-line-derived neurotrophic factor GDNF is considered to be a potential therapeutic agent for neurons and treatment for neurodegenerative diseases [212, 235, 236]. For example, an implant loaded with

132

Vector

Biomaterial

Physical state

Gene therapy

Biomaterial/tissue integration

References

Retrovirus and LV

Natural

–

–

–

–

–

Synthetic

PES

Membrane

Indirect

No

[212, 213]

Plasmid

Natural

Alginate

Microspheres

Indirect

No

[214, 215]

Synthetic

PAN-PVC

Fibers

Indirect

No

[216–223]

Polysulfone

Membrane

Indirect

No

[224–227]

PES

Membrane

Indirect

No

[228–232]

PVA

Scaffold

Indirect

No

[233, 234]

4.  Bioengineering strategies for gene delivery

TABLE 4.3  Neurodegenerative disease therapeutic strategies utilizing biomaterial carriers and gene therapies



5  Gene delivery via biomaterial strategies

133

­ uman cells modified via LV to express localized therapeutic levels of h GDNF have been tested as a potential therapeutic strategy for epileptic seizures. Modified human cells encoding (GDNF) loaded into polyethersulfone (PES) membranes implanted in the hippocampus of rats showed suppressed recurrent generalized seizures when compared to empty the PES membrane control [212]. However, cell damage and inflammation were not significantly different between the groups [212]. In contrast, human fibroblasts modified with LV encoding GDNF and seeded inside PVA and encapsulated in PES membrane caused statistically significant behavioral and localized neurostructural improvements in a striatal rat model of Parkinson’s disease [213]. Furthermore, these improvements persisted for the duration of the 6-week follow-up postimplant removal from then striatum [213]. 5.2.2 Nonviral Indirect gene therapies utilizing nonviral genetic modification of cells and biomaterial delivery have been extensively studied. All have utilized implantable biomaterials encapsulating modified cells to allow for localized therapeutic secretion of biomolecules directly to the CNS—therefore surpassing the BBB limitation. This method has demonstrated effectiveness over a broad range of biomolecules and has resulted in several clinical protocols and trials [230, 232, 233, 237]. β-Glucuronidase is a lysosomal enzyme, and if deficient, can cause CNS abnormalities [215]. pDNA carrying β-glucuronidase transfected into mouse fibroblasts which were then encapsulated into alginate microbeads resulted in significant reductions in both lysosomal accumulations and behavior anomalies in a beta-glucuronidase-deficient mouse model. The majority of biomaterial encapsulated indirect gene therapies focus on the delivery of growth factors. Encapsulated GDNF pDNA transfected cells have shown significant benefit in rat models of Parkinson’s disease [224, 226]. GDNF pDNA transfected baby hamster kidney (BHK) cells infused into polysulfone capsules showed significant nigrostriatal dopaminergic fibers and cell bodies localized to the striatum implant site compared to encapsulated unmodified BHK cell controls 6 months postimplantation [224]. In addition, GDNF pDNA modified cells encapsulated in PES ant applied to the striatum also demonstrated that earlier therapeutic intervention in Parkinson’s resulted in significantly higher physiological preservation and significant reductions in behavioral anomalies [226]. Some neurodegenerative diseases have comorbidity with cerebrovascular diseases, atherosclerosis, and cerebral microvascular pathology [238–240]. Due to this correlation, VEGF has been the focus of indirect nonviral gene therapies to promote new vessel formation in the brain to clear detrimental deposits [214]. VEGF pDNA transfected BHK fibroblasts that were encapsulated into alginate microbeads, which were then into

134

4.  Bioengineering strategies for gene delivery

the craniotomy of Alzheimer’s disease mutant mice [214]. Significant vessel growth was seen 2 weeks and 3 months post application and significant AB burden was decreased was also seen. In addition, significant reductions in apoptotic death were seen in the VEGF microcapsule-treated group and significant benefits in behavioral activity and retention. In addition, VEGF has also shown the ability to act as a neuroprotective agent for neurological disorders and has shown promise as a potent neurorescue molecule [214, 225, 227]. Parkinson’s model rats implanted in the striatum with polysulfone capsule loaded with pDNA VEGF modified BHK cells also showed neuroprotection in both neuronal and glial cell lines and significant behavioral improvements 2  months following implantation relative to controls [225]. Similarly, VEGF modified BHK cells encapsulated in polysulfone capsules showed significant increases in not only neuron preservation, but also glial cell proliferation localized to the striatum implant site [227]. One of the most well-documented growth factors studied in biomaterial encapsulated indirect gene therapies is a ciliary neurotrophic factor (CNTF). CNTF is a neuroprotective cytokine that has shown to protect striatal neurons in multiple Huntington’s disease animal models [221, 222, 229]. Many studies have focused on the implantation of devices carrying indirect CNTF gene therapies, including clinical protocols for the treatment of Huntington’s disease and ALS [228, 231]. CNTF modified BHK cells loaded into poly(acrylonitrile-vinylchloride) (PAN-PVC) copolymer fibers and implanted in Huntington’s disease model rat lateral ventricle showed significant beneficial behavioral changes and decreases in brain lesion size [221]. Similarly, CNTF modified BKH cells loaded into PANPVC fiber implants placed in the intrastrial of cynomolgus monkeys to measure CNTF protective effects against acid injection Huntington’s disease model showed significant protective results in BHK-CNTF-treated groups compared to CNTF-negative scaffold groups [222]. The striata of Huntington’s disease model rats implanted with PES membranes loaded with CNTF loaded collagen suspended BHK cells resulted in significant neural protection in the CNTF positive group compared to untreated lesioned controls [229]. This gene therapy and biomaterial delivery strategy have been the focus of a treatment ALS clinical study. BHK-CNTF introduced into a collagen filled PES membrane and implanted into the lumbar intrathecal space of six early stage ALS patients [232]. Implants were removed after 3 months, the patients were evaluated, and secondary implants were introduced for 6  months [232]. Despite consistent CNTF secretion from the implants, clinical evaluations determined disease progression continued in all six patients according to ALS clinical rating scales after implantation [232]. Similarly, a phase 1 clinical study of six stage 1 or stage 2 Huntington’s disease patients was treated with BHK line expressing CNTF encapsulated within a polysulfone membrane for 2 years



5  Gene delivery via biomaterial strategies

135

(new implants were given every 6  months) [230]. Overall, there was no statistically significant clinical benefit attributed to intracerebral release of CNTF from any of the six subjects based on neurological, neuropsychological, and motor assessments [230]. However, safety, feasibility, and tolerability of this therapeutic option were demonstrated. One limitation of this phase I study was that only a single capsule at a time was placed into patients when preclinical primate models showed that four implants were needed for statistically significant results to be returned [229]. In addition, cell survival within these capsules varied upon retrieval and 13 of the 24 total capsules failed to release significant quantities of CNTF [230]. Nerve growth factor (NGF) is the most studied growth factor utilized in indirect gene therapies for the study of neurodegenerative diseases. NGF has shown to support ganglia viability and, consequently, it has been studied in a variety of animal models to treat neurodegenerative disorders, specifically Huntington’s and Alzheimer’s disease [216–219]. NGF pDNA transfected fibroblasts were mixed with collagen and loaded into fibers of poly(acrylonitrile-co-vinyl chloride) (PAN-PVC) copolymer before implantation into rat caudoputamen for potential therapeutic benefits for Alzheimer’s disease [216]. The NGF cell loaded implant group had significantly increased neurodensity compared to the empty implant control [216]. Similar fimbria-fornix lesioned rat models for Alzheimer’s returned statistically significant axotomized medial septal cholinergic neurons compared to NGF-deficient controls [218]. Rat fibroblasts transfected with pDNA encoding NGF loaded into PAN-PVC and placed into aspirative lesions present in the fimbria and dorsal fornix of rats [223]. The encapsulated NGF expressing cell group showed prevention of ­lesion-induced loss of gene expression not seen in the empty implant control [223]. Behavioral benefits have also been documented in rat models of neurodegenerative disease. Rats modeling extensive neurodegenerative damage via acid injections were exposed to BKH-NGF secreting cells encapsulated in PAN-PVC, and as a result significant behavior changes [220]. Similar results were seen in monkey models for Alzheimer’s disease upon PAN-PVC implantation of NGF modified BHK cells into the lateral ventricles [217]. In addition to attenuated neuron loss, robust sprouting of cholinergic fibers was observed in the adjacent lateral septum [217]. Rhesus monkeys were treated with BHK cells transfected with NGF loaded into PAN-PVC to look at potential benefits for Alzheimer’s [219]. Lesion-induced degeneration of sepal neurons was significantly decreased in BHK-NGF-treated cells compared to NGF-deficient controls [219]. Biomaterial delivered indirect gene therapies utilizing NGF have also been the focus of possible clinical applications, and to date has been the focus of a clinical trial. The first indirect gene therapy phase 1 clinical trial utilizing PVA scaffold containing genetically modified human cells expressing NGF applied to the basal forebrain for the purpose of

136

4.  Bioengineering strategies for gene delivery

halting the degeneration of cholinergic neurons and cognitive decline in Alzheimer’s patients. While no inflammation or device displacement was seen, no statistically significant increases in cognition scores were seen despite low persistent NGF secretion was detected in half of the six patients tested at 1-year postimplantation [233, 234].

6  Current therapeutic outlook Gene therapies have remarkable therapeutic potential for both tissue regeneration and degenerative disease treatment due to their ability to localize and continuously produce bioactive levels of therapeutic molecules. Biomaterial systems used in tandem with gene therapies offer enhanced safety profiles, predictable dosing, and increased therapeutic efficiency [4, 7, 11]. Biomaterials can be utilized as controlled delivery devices for gene therapy vectors and genetically modified cells, or as scaffolds that promote cell adhesion and infiltration for tissue integration in tissue engineering endeavors. Gene therapies currently on the market largely rely on ex vivo manipulation, which has several limitations including the limited supply of donor cells and the complexity and high costs associated with ex vivo culture and manipulation [4, 7]. Therefore, expansion in the field of gene therapy relies on safe and efficient methods of introducing gene therapy vectors in situ. Future development of genetic elements to create reliably inducible vector constructs will increase external control of therapeutic expression of gene cassettes, and advances in viral pseudotyping will increase transduction efficiency of targeted cell types. In addition to these vector developments, advancements in biomaterial carriers of these refined vectors will further increase localization, presentation, and survival of not only vectors but modified cells as well.

References [1] Genetics Home Reference. What is gene therapy? Genetics Home Reference. Available from: https://ghr.nlm.nih.gov/primer/therapy/genetherapy. [2] Ginn SL, Amaya AK, Alexander IE, Edelstein M, Abedi MR. Gene therapy clinical trials worldwide to 2017: an update. J Gene Med 2018;20(5):e3015. [3] Naldini L. Gene therapy returns to centre stage. Nature 2015;526(7573):351–60. [4] Williams  PA, Campbell  KT, Silva  EA. Biomaterials and cells for revascularization. In: Emerich DF, Orive G, editors. Cell therapy: Current status and future directions. Molecular and translational medicineCham: Springer International Publishing; 2017. p. 139–72. [5] Lantz M, Malik S, Slevin ML, Olsson I. Infusion of tumor necrosis factor (TNF) causes an increase in circulating TNF-binding protein in humans. Cytokine 1990;2(6):402–6. [6] Annex  BH. Therapeutic angiogenesis for critical limb ischaemia. Nat Rev Cardiol 2013;10(7):387–96. [7] Madrigal JL, Stilhano R, Silva EA. Biomaterial-guided gene delivery for musculoskeletal tissue repair. Tissue Eng B Rev 2017;23(4):347–61.



References

137

[8] Howarth JL, Lee YB, Uney JB. Using viral vectors as gene transfer tools (cell biology and toxicology special issue: ETCS-UK 1  day meeting on genetic manipulation of cells). Cell Biol Toxicol 2010;26(1):1–20. [9] Li  J, Mooney  DJ. Designing hydrogels for controlled drug delivery. Nat Rev Mater 2016;1(12):16071. [10] Delaittre G, Greiner AM, Pauloehrl T, Bastmeyer M, Barner-Kowollik C. Chemical approaches to synthetic polymer surface biofunctionalization for targeted cell adhesion using small binding motifs. Soft Matter 2012;8(28):7323–47. [11] Sailaja  G, HogenEsch  H, North  A, Hays  J, Mittal  SK. Encapsulation of recombinant adenovirus into alginate microspheres circumvents vector specific immune response. Gene Ther 2002;9(24):1722–9. [12] Bryant  LM, Christopher  DM, Giles  AR, Hinderer  C, Rodriguez  JL, Smith  JB, et  al. Lessons learned from the clinical development and market authorization of glybera. Hum Gene Ther Clin Dev 2013;24(2):55–64. [13] Touchot  N, Flume  M. Early insights from commercialization of gene therapies in Europe. Genes 2017;8(2):78. [14] Pol J, Buqué A, Aranda F, Bloy N, Cremer I, Eggermont A, et al. Trial watch—oncolytic viruses and cancer therapy. OncoImmunology 2016;5(2):e1117740. [15] Andtbacka  RHI, Kaufman  HL, Collichio  F, Amatruda  T, Senzer  N, Chesney  J, et  al. Talimogene laherparepvec improves durable response rate in patients with advanced melanoma. J Clin Oncol 2015;33(25):2780–8. [16] Bordignon C, Notarangelo LD, Nobili N, Ferrari G, Casorati G, Panina P, et al. Gene therapy in peripheral blood lymphocytes and bone marrow for ADA-immunodeficient patients. Science 1995;270(5235):470–5. [17] South  E, Cox  E, Meader  N, Woolacott  N, Griffin  S. Strimvelis® for treating severe combined immunodeficiency caused by adenosine deaminase deficiency: an evidence review group perspective of a NICE highly specialised technology evaluation. PharmacoEconomics 2019;3(2):151–61. [18] Maude  SL, Laetsch  TW, Buechner  J, Rives  S, Boyer  M, Bittencourt  H, et  al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med 2018;378(5):439–48. [19] Shah B, Huynh V, Sender LS, Lee DW, Castro JE, Wierda WG, et al. High rates of minimal residual disease-negative (MRD−) complete responses (CR) in adult and pediatric and patients with relapsed/refractory acute lymphoblastic leukemia (R/R ALL) treated with KTE-C19 (anti-CD19 chimeric antigen receptor [CAR] T cells): preliminary results of the ZUMA-3 and ZUMA-4 trials. Blood 2016;128(22):2803. [20] Gardner R, Leger KJ, Annesley CE, Summers C, Rivers J, Gust J, et al. Decreased rates of severe CRS seen with early intervention strategies for CD19 CAR-T cell toxicity management. Blood 2016;128(22):586. [21] Neelapu  SS, Locke  FL, Bartlett  NL, Lekakis  LJ, Miklos  DB, Jacobson  CA, et  al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med 2017;377(26):2531–44. [22] Smalley  E. First AAV gene therapy poised for landmark approval. Nat Biotechnol 2017;35:998–9. [23] Russell  S, Bennett  J, Wellman  JA, Chung  DC, Yu  Z-F, Tillman  A, et  al. Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial. The Lancet 2017;390(10097):849–60. [23a] Mendell  JR, Al-Zaidy  S, Shell  R, Arnold  WD, Rodino-Klapac  LR, Prior  TW, et  al. Single-dose gene-replacement therapy for spinal muscular atrophy. N Engl J Med 2017;377(18):1713–22. [24] Senior M. After Glybera’s withdrawal, what’s next for gene therapy? Nat Biotechnol 2017;35:491–2.

138

4.  Bioengineering strategies for gene delivery

[25] Abou-El-Enein M, Elsanhoury A, Reinke P. Overcoming challenges facing advanced therapies in the EU market. Cell Stem Cell 2016;19(3):293–7. [26] Xie  F. Highly priced gene therapies: a wake-up call for early price regulation. Pharmacoecon Open 2018;36(8):883–8. [27] Whittington  MD, McQueen  RB, Campbell  JD. Considerations for cost-effectiveness analysis of curative pediatric therapies. JAMA Pediatr 2018;172(5):409–10. [28] Rich  K, Terry  SF. The price of precision: genetic testing and drug costs in America. Genet Test Mol Biomarkers 2018;22(7):403–4. [28a] Keeler  AM, Flotte  TR. Recombinant adeno-associated virus gene therapy in light of Luxturna (and Zolgensma and Glybera): where are we, and how did we get here? Annu Rev Virol 2019;6(1):22.1–21. [29] Edelstein ML, Abedi MR, Wixon J, Edelstein RM. Gene therapy clinical trials worldwide 1989–2004—an overview. J Gene Med 2004;6(6):597–602. [30] Edelstein ML, Abedi MR, Wixon J. Gene therapy clinical trials worldwide to 2007—an update. J Gene Med 2007;9(10):833–42. [31] Search of: Active, not recruiting Studies | gene therapy | Phase 3, 4—List Results— ClinicalTrials.gov. [32] Kumru OS, Wang Y, Gombotz CWR, Kelley-Clarke B, Cieplak W, Kim T, et al. Physical characterization and stabilization of a lentiviral vector against adsorption and freezethaw. J Pharm Sci 2018;107(11):2764–74. [33] Bryson  PD, Wang  P. Lentivector vaccines. In: Lattime  EC, Gerson  SL, editors. Gene ­therapy of cancer. 3rd ed. San Diego, CA: Academic Press; 2014. p. 345–61. [cited 2019 Jan 28]. [chapter 24]. [34] Croyle  MA, Callahan  SM, Auricchio  A, Schumer  G, Linse  KD, Wilson  JM, et  al. PEGylation of a vesicular stomatitis virus G pseudotyped lentivirus vector prevents inactivation in serum. J Virol 2004;78(2):912–21. [35] Stilhano RS, Madrigal JL, Wong K, Williams PA, Martin PKM, Yamaguchi FSM, et al. Injectable alginate hydrogel for enhanced spatiotemporal control of lentivector delivery in murine skeletal muscle. J Control Release 2016;237:42–9. [36] Anderson WF. Prospects for human gene therapy. Science 1984;226(4673):401–9. [37] Appaiahgari  MB, Vrati  S. Adenoviruses as gene/vaccine delivery vectors: promises and pitfalls. Expert Opin Biol Ther 2015;15(3):337–51. [38] Cotten  M, Weber  JM. The adenovirus protease is required for virus entry into host cells. Virology 1995;213(2):494–502. [39] Nemerow GR, Stewart PL, Reddy VS. Structure of human adenovirus. Curr Opin Virol 2012;2(2):115–21. [40] Parks RJ, Graham FL. A helper-dependent system for adenovirus vector production helps define a lower limit for efficient DNA packaging. J Virol 1997;71:6. [41] Stratford-Perricaudet LD, Levrero M, Chasse J-F, Perricaudet M, Briand P. Evaluation of the transfer and expression in mice of an enzyme-encoding gene using a human adenovirus vector. Hum Gene Ther 1990;1(3):241–56. [42] Lai CM, Lai YKY, Rakoczy PE. Adenovirus and adeno-associated virus vectors. DNA Cell Biol 2002;21(12):895–913. [43] Berns KI, Giraud C. Biology of adeno-associated virus. In: Berns KI, Giraud C, editors. Adeno-associated virus (AAV) vectors in gene therapy. Current topics in microbiology and immunologyBerlin/Heidelberg: Springer; 1996. p. 1–23. [44] Naso  MF, Tomkowicz  B, Perry  WL, Strohl  WR. Adeno-associated virus (AAV) as a vector for gene therapy. BioDrugs 2017;31(4):317–34. [45] Monahan PE, Samulski RJ. Adeno-associated virus vectors for gene therapy: more pros than cons? Mol Med Today 2000;6(11):433–40. [46] Wright JF, Le T, Prado J, Bahr-Davidson J, Smith PH, Zhen Z, et al. Identification of factors that contribute to recombinant AAV2 particle aggregation and methods to prevent its occurrence during vector purification and formulation. Mol Ther 2005;12(1):171–8.



References

139

[47] Hermonat PL, Muzyczka N. Use of adeno-associated virus as a mammalian DNA cloning vector: transduction of neomycin resistance into mammalian tissue culture cells. Proc Natl Acad Sci U S A 1984;81(20):6466–70. [48] Shen  H, Hu  Y, Saltzman  WM. DNA diffusion in mucus: effect of size, topology of DNAs, and transfection reagents. Biophys J 2006;91(2):639–44. [49] Kretzmann  JA, Ho  D, Evans  CW, Plani-Lam  JHC, Garcia-Bloj  B, Elaaf Mohamed  A, et al. Synthetically controlling dendrimer flexibility improves delivery of large plasmid DNA. Chem Sci 2017;8(4):2923–30. [50] Bertoni C, Jarrahian S, Wheeler TM, Li Y, Olivares EC, Calos MP, et al. Enhancement of plasmid-mediated gene therapy for muscular dystrophy by directed plasmid integration. Proc Natl Acad Sci 2006;103(2):419–24. [51] Ledley FD. Nonviral gene therapy: the promise of genes as pharmaceutical products. Hum Gene Ther 1995;6(9):1129–44. [52] Baxter JD. Recombinant DNA and medical progress. Hosp Pract 1980;15(2):57–67. [53] Sakuma  T, Barry  MA, Ikeda  Y. Lentiviral vectors: basic to translational. Biochem J 2012;443(3):603–18. [54] Collins M, Thrasher A. Gene therapy: progress and predictions. Proc R Soc B Biol Sci 2015;282(1821). [55] Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, et al. A third-generation lentivirus vector with a conditional packaging system. J Virol 1998;72:9. [56] Naldini L, Blömer U, Gallay P, Ory D, Mulligan R, Gage FH, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 1996;272(5259):263–7. [57] Page  KA, Landau  NR, Littman  DR. Construction and use of a human immunodeficiency virus vector for analysis of virus infectivity. J Virol 1990;64(11):5270–6. [58] Yu SF, von Ruden T, Kantoff PW, Garber C, Seiberg M, Ruther U, et al. Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells. Proc Natl Acad Sci 1986;83(10):3194–8. [59] Freed  EO, Martin  MA. HIV-1 infection of non-dividing cells. Nature 1994;369 (6476):107–8. [60] Bukrinsky  MI, Haggerty  S, Dempsey  MP, Sharova  N, Adzhubei  A, Spitz  L, et  al. A nuclear localization signal within HIV-1 matrix protein that governs infection of non-­ dividing cells. Nature 1993;365(6447):666–9. [61] Sheridan C. Gene therapy finds its niche. Nat Biotechnol 2011;29:121–8. [62] Schröder ARW, Shinn P, Chen H, Berry C, Ecker JR, Bushman F. HIV-1 integration in the human genome favors active genes and local hotspots. Cell 2002;110(4):521–9. [63] Mitchell RS, Beitzel BF, Schroder ARW, Shinn P, Chen H, Berry CC, et al. Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences. Michael Emerman, editor PLoS Biol 2004;2(8):e234. [64] Wang GP, Ciuffi A, Leipzig J, Berry CC, Bushman FD. HIV integration site selection: analysis by massively parallel pyrosequencing reveals association with epigenetic modifications. Genome Res 2007;17(8):1186–94. [65] Pannell D, Ellis J. Silencing of gene expression: implications for design of retrovirus vectors. Rev Med Virol 2001;11(4):205–17. [66] Chira S, Jackson CS, Oprea I, Ozturk F, Pepper MS, Diaconu I, et al. Progresses towards safe and efficient gene therapy vectors. Oncotarget 2015;6(31):30675–703. [67] Stewart PL, Burnett RM, Cyrklaff M, Fuller SD. Image reconstruction reveals the complex molecular organization of adenovirus. Cell 1991;67(1):145–54. [68] Waehler R, Russell SJ, Curiel DT. Engineering targeted viral vectors for gene therapy. Nat Rev Genet 2007;8(8):573–87. [69] Leen AM, Christin A, Khalil M, Weiss H, Gee AP, Brenner MK, et al. Identification of hexon-specific CD4 and CD8 T-cell epitopes for vaccine and immunotherapy. J Virol 2008;82(1):546–54.

140

4.  Bioengineering strategies for gene delivery

[70] Choi J-W, Kang E, Kwon O-J, Yun TJ, Park H-K, Kim P-H, et al. Local sustained delivery of oncolytic adenovirus with injectable alginate gel for cancer virotherapy. Gene Ther 2013;20(9):880–92. [71] Branca  MA. Gene therapy: cursed or inching towards credibility? Nat Biotechnol 2005;23:519–21. [72] Kalyanasundaram  S, Feinstein  S, Nicholson  JP, Leong Jr. KW. RIG. Coacervate microspheres as carriers of recombinant adenoviruses. Cancer Gene Ther 1999;6(2):107–12. [73] Srivastava  A, Lusby  EW, Berns  KI. Nucleotide sequence and organization of the ­adeno-associated virus 2 genome. J Virol 1983;45(2):555–64. [74] Samulski RJ, Muzyczka N. AAV-mediated gene therapy for research and therapeutic purposes. Annu Rev Virol 2014;1(1):427–51. [75] DiMattia MA, Nam H-J, Vliet KV, Mitchell M, Bennett A, Gurda BL, et al. Structural insight into the unique properties of adeno-associated virus serotype 9. J Virol 2012;86(12):6947–58. [76] Im  D-S, Muzyczka  N. The AAV origin binding protein Rep68 is an ATP-dependent site-specific endonuclease with DNA helicase activity. Cell 1990;61(3):447–57. [77] King  JA, Dubielzig  R, Grimm  D, Kleinschmidt  JA. DNA helicase-mediated packaging of adeno-associated virus type 2 genomes into preformed capsids. EMBO J 2001;20(12):3282–91. [78] Im DS, Muzyczka N. Partial purification of adeno-associated virus Rep78, Rep52, and Rep40 and their biochemical characterization. J Virol 1992;66(2):1119–28. [79] Sonntag  F, Bleker  S, Leuchs  B, Fischer  R, Kleinschmidt  JA. Adeno-associated virus type 2 capsids with externalized VP1/VP2 trafficking domains are generated prior to ­passage through the cytoplasm and are maintained until uncoating occurs in the nucleus. J Virol 2006;80(22):11040–54. [80] Sonntag F, Schmidt K, Kleinschmidt JA. A viral assembly factor promotes AAV2 capsid formation in the nucleolus. Proc Natl Acad Sci U S A 2010;6:201001673. [81] Grieger  JC, Snowdy  S, Samulski  RJ. Separate basic region motifs within the ­adeno-associated virus capsid proteins are essential for infectivity and assembly. J Virol 2006;80(11):5199–210. [82] Wu  Z, Asokan  A, Samulski  RJ. Adeno-associated virus serotypes: vector toolkit for human gene therapy. Mol Ther 2006;14(3):316–27. [83] Flannery  JG, Zolotukhin  S, Vaquero  MI, LaVail  MM, Muzyczka  N, Hauswirth  WW. Efficient photoreceptor-targeted gene expression in  vivo by recombinant adeno-­ associated virus. Proc Natl Acad Sci U S A 1997;94(13):6916–21. [84] Peel AL, Zolotukhin S, Schrimsher GW, Muzyczka N, Reier PJ. Efficient transduction of green fluorescent protein in spinal cord neurons using adeno-associated virus vectors containing cell type-specific promoters. Gene Ther 1997;4(1):16–24. [85] Snyder RO, Spratt SK, Lagarde C, Bohl D, Kaspar B, Sloan B, et al. Efficient and stable adeno-associated virus-mediated transduction in the skeletal muscle of adult immunocompetent mice. Hum Gene Ther 1997;8(16):1891–900. [86] Acland GM, Aguirre GD, Bennett J, Aleman TS, Cideciyan AV, Bennicelli J, et al. Longterm restoration of rod and cone vision by single dose rAAV-mediated gene transfer to the retina in a canine model of childhood blindness. Mol Ther 2005;12(6):1072–82. [87] Lisowski L, Tay SS, Alexander IE. Adeno-associated virus serotypes for gene therapeutics. Curr Opin Pharmacol 2015;24:59–67. [88] Boutin S, Monteilhet V, Veron P, Leborgne C, Benveniste O, Montus MF, et al. Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors. Hum Gene Ther 2010;21(6):704–12. [89] Rey-Rico A, Cucchiarini M. Controlled release strategies for rAAV-mediated gene delivery. Acta Biomater 2016;29:1–10.



References

141

[90] Prazeres DMF, Monteiro GA. Plasmid biopharmaceuticals. Microbiol Spectr 2014;2(6). [cited 2018 Dec 19]. Available from: http://www.asmscience.org/content/journal/ microbiolspec/10.1128/microbiolspec.PLAS-0022-2014. [91] Aronovich EL, Bell JB, Belur LR, Gunther R, Koniar B, Erickson DCC, et al. Prolonged expression of a lysosomal enzyme in mouse liver after sleeping beauty transposon-­ mediated gene delivery: implications for non-viral gene therapy of mucopolysaccharidoses. J Gene Med 2007;9(5):403–15. [92] Chen Z-Y, He C-Y, Kay MA. Improved production and purification of minicircle DNA vector free of plasmid bacterial sequences and capable of persistent transgene expression in vivo. Hum Gene Ther 2005;16(1):126–31. [93] Osborn MJ, McElmurry RT, Lees CJ, DeFeo AP, Chen Z-Y, Kay MA, et al. Minicircle DNA-based gene therapy coupled with immune modulation permits long-term expression of α-l-iduronidase in mice with mucopolysaccharidosis type I. Mol Ther 2011;19(3):450–60. [94] Ehrhardt A, Xu H, Huang Z, Engler JA, Kay MA. A direct comparison of two nonviral gene therapy vectors for somatic integration: in vivo evaluation of the bacteriophage integrase ϕC31 and the sleeping beauty transposase. Mol Ther 2005;11(5):695–706. [95] Torio-Padron  N, Borges  J, Momeni  A, Mueller  MC, Tegtmeier  FT, Bjoern Stark  G. Implantation of VEGF transfected preadipocytes improves vascularization of fibrin implants on the cylinder chorioallantoic membrane (CAM) model. Minim Invasive Ther Allied Technol 2007;16(3):155–62. [96] Zu Y, Huang S, Liao W-C, Lu Y, Wang S. Gold nanoparticles enhanced electroporation for mammalian cell transfection. J Biomed Nanotechnol 2014;10(6):982–92. [97] Kimelman-Bleich  N, Pelled  G, Zilberman  Y, Kallai  I, Mizrahi  O, Tawackoli  W, et  al. Targeted gene-and-host progenitor cell therapy for nonunion bone fracture repair. Mol Ther 2011;19(1):53–9. [98] Nomikou N, Feichtinger GA, Redl H, McHale AP. Ultrasound-mediated gene transfer (sonoporation) in fibrin-based matrices: potential for use in tissue regeneration. J Tissue Eng Regen Med 2016;10(1):29–39. [99] Helary  C, Desimone  M. Recent advances in biomaterials for tissue engineering and controlled drug delivery. Curr Pharm Biotechnol 2015;16(7):635–45. [100] Hortensius RA, Harley BA. Naturally derived biomaterials for addressing inflammation in tissue regeneration. Exp Biol Med 2016;241(10):1015–24. [101] Kim JK, Kim HJ, Chung J-Y, Lee J-H, Young S-B, Kim Y-H. Natural and synthetic biomaterials for controlled drug delivery. Arch Pharm Res 2014;37(1):60–8. [102] Lee EJ, Kasper FK, Mikos AG. Biomaterials for tissue engineering. Ann Biomed Eng 2014;42(2):323–37. [103] Shin  H, Jo  S, Mikos  AG. Biomimetic materials for tissue engineering. Biomaterials 2003;24(24):4353–64. [104] Suk  JS, Xu  Q, Kim  N, Hanes  J, Ensign  LM. PEGylation as a strategy for improving nanoparticle-based drug and gene delivery. Adv Drug Deliv Rev 2016;99:28–51. [105] Yao H, Chen S-C, Shen Z, Huang Y-C, Zhu X, Wang X, et al. Functional characterization of a PEI-CyD-FA-coated adenovirus as delivery vector for gene therapy. Curr Med Chem 2013;20(20):2601–8. [106] Rezaee  M, Oskuee  RK, Nassirli  H, Malaekeh-Nikouei  B. Progress in the development of lipopolyplexes as efficient non-viral gene delivery systems. J Control Release 2016;236:1–14. [107] Yin H, Kanasty RL, Eltoukhy AA, Vegas AJ, Dorkin JR, Anderson DG. Non-viral vectors for gene-based therapy. Nat Rev Genet 2014;15(8):541–55. [108] Jones CH, Chen C-K, Ravikrishnan A, Rane S, Pfeifer BA. Overcoming nonviral gene delivery barriers: perspective and future. Mol Pharm 2013;10(11):4082–98. [109] Zylberberg C, Gaskill K, Pasley S, Matosevic S. Engineering liposomal nanoparticles for targeted gene therapy. Gene Ther 2017;24(8):441–52.

142

4.  Bioengineering strategies for gene delivery

[110] Kim J, Wilson DR, Zamboni CG, Green JJ. Targeted polymeric nanoparticles for cancer gene therapy. J Drug Target 2015;23(7–8):627–41. [111] Brunger JM, Huynh NPT, Guenther CM, Perez-Pinera P, Moutos FT, Sanchez-Adams J, et al. Scaffold-mediated lentiviral transduction for functional tissue engineering of cartilage. Proc Natl Acad Sci 2014;111(9):E798–806. [112] De Laporte L, Cruz Rea J, Shea LD. Design of modular non-viral gene therapy vectors. Biomaterials 2006;27(7):947–54. [113] Jang J-H, Schaffer DV, Shea LD. Engineering biomaterial systems to enhance viral vector gene delivery. Mol Ther 2011;19(8):1407–15. [114] Drury JL, Mooney DJ. Hydrogels for tissue engineering: scaffold design variables and applications. Biomaterials 2003;24(24):4337–51. [115] Knop  K, Hoogenboom  R, Fischer  D, Schubert  US. Poly(ethylene glycol) in drug delivery: pros and cons as well as potential alternatives. Angew Chem Int Ed 2010;49(36):6288–308. [116] Harris JM. Poly(ethylene glycol) chemistry: Biotechnical and biomedical applications. New York City: Springer Science & Business Media; 2013. 395 p. [117] Yoo HS, Park TG. Biodegradable polymeric micelles composed of doxorubicin conjugated PLGA–PEG block copolymer. J Control Release 2001;70(1):63–70. [118] Thomas AM, Gomez AJ, Palma JL, Yap WT, Shea LD. Heparin–chitosan nanoparticle functionalization of porous poly(ethylene glycol) hydrogels for localized lentivirus delivery of angiogenic factors. Biomaterials 2014;35(30):8687–93. [119] Shepard JA, Virani FR, Goodman AG, Gossett TD, Shin S, Shea LD. Hydrogel macroporosity and the prolongation of transgene expression and the enhancement of angiogenesis. Biomaterials 2012;33(30):7412–21. [120] Quick  DJ, Anseth  KS. DNA delivery from photocrosslinked PEG hydrogels: encapsulation efficiency, release profiles, and DNA quality. J Control Release 2004;96(2):341–51. [121] Luu YK, Kim K, Hsiao BS, Chu B, Hadjiargyrou M. Development of a nanostructured DNA delivery scaffold via electrospinning of PLGA and PLA–PEG block copolymers. J Control Release 2003;89(2):341–53. [122] West JL, Hubbell JA. Polymeric biomaterials with degradation sites for proteases involved in cell migration. Macromolecules 1999;32(1):241–4. [123] Zisch AH, Lutolf MP, Ehrbar M, Raeber GP, Rizzi SC, Davies N, et al. Cell-demanded release of VEGF from synthetic, biointeractive cell ingrowth matrices for vascularized tissue growth. FASEB J 2003;17(15):2260–2. [124] Moon CY, Choi J-W, Kasala D, Jung S-J, Kim SW, Yun C-O. Dual tumor targeting with pH-sensitive and bioreducible polymer-complexed oncolytic adenovirus. Biomaterials 2015;41:53–68. [125] Yao X, Yoshioka Y, Morishige T, Eto Y, Watanabe H, Okada Y, et al. Systemic administration of a PEGylated adenovirus vector with a cancer-specific promoter is effective in a mouse model of metastasis. Gene Ther 2009;16(12):1395–404. [126] Fan G, Fan M, Wang Q, Jiang J, Wan Y, Gong T, et al. Bio-inspired polymer envelopes around adenoviral vectors to reduce immunogenicity and improve in  vivo kinetics. Acta Biomater 2016;30:94–105. [127] Leggiero  E, Astone  D, Cerullo  V, Lombardo  B, Mazzaccara  C, Labruna  G, et  al. PEGylated helper-dependent adenoviral vector expressing human Apo A-I for gene therapy in LDLR-deficient mice. Gene Ther 2013;20(12):1124–30. [128] Kichler  A. Gene transfer with modified polyethylenimines. J Gene Med 2004;6(S1) :S3–10. [129] Kim B-S, Mooney DJ. Development of biocompatible synthetic extracellular matrices for tissue engineering. Trends Biotechnol 1998;16(5):224–30. [130] Makadia HK, Siegel SJ. Poly lactic-co-glycolic acid (PLGA) as biodegradable controlled drug delivery carrier. Polymers 2011;3(3):1377–97.



References

143

[131] Göpferich  A. Mechanisms of polymer degradation and erosion. Biomaterials 1996;17(2):103–14. [132] Shea LD, Smiley E, Bonadio J, Mooney DJ. DNA delivery from polymer matrices for tissue engineering. Nat Biotechnol 1999;17(6):551–4. [133] Li  J, Xu  Q, Teng  B, Yu  C, Li  J, Song  L, et  al. Investigation of angiogenesis in bioactive 3-dimensional poly(d,l-lactide-co-glycolide)/nano-hydroxyapatite scaffolds by in vivo multiphoton microscopy in murine calvarial critical bone defect. Acta Biomater 2016;42:389–99. [134] Jang J-H, Rives CB, Shea LD. Plasmid delivery in vivo from porous tissue-engineering scaffolds: transgene expression and cellular transfection. Mol Ther 2005;12(3):475–83. [135] Ito Y. Covalently immobilized biosignal molecule materials for tissue engineering. Soft Matter 2008;4(1):46–56. [136] Jin Yoon  J, Ho Song  S, Sung Lee  D, Park  TG. Immobilization of cell adhesive RGD peptide onto the surface of highly porous biodegradable polymer scaffolds fabricated by a gas foaming/salt leaching method. Biomaterials 2004;25(25):5613–20. [137] Jabbarzadeh  E, Starnes  T, Khan  YM, Jiang  T, Wirtel  AJ, Deng  M, et  al. Induction of angiogenesis in tissue-engineered scaffolds designed for bone repair: a combined gene ­therapy-cell transplantation approach. Proc Natl Acad Sci U S A 2008;105(32):11099–104. [138] Duan C, Liu J, Yuan Z, Meng G, Yang X, Jia S, et al. Adenovirus-mediated transfer of VEGF into marrow stromal cells combined with PLGA/TCP scaffold increases vascularization and promotes bone repair in vivo. Arch Med Sci 2014;10(1):174–81. [139] Hidaka C, Ibarra C, Hannafin JA, Torzilli PA, Quitoriano M, Jen S-S, et al. Formation of vascularized meniscal tissue by combining gene therapy with tissue engineering. Tissue Eng 2002;8(1):93–105. [140] Smidsrød  O, Skjåk-Braek  G. Alginate as immobilization matrix for cells. Trends Biotechnol 1990;8:71–8. [141] Silva EA, Mooney DJ. Spatiotemporal control of vascular endothelial growth factor delivery from injectable hydrogels enhances angiogenesis. J Thromb Haemost 2007;5(3):590–8. [142] Hao X, Silva EA, Månsson-Broberg A, Grinnemo K-H, Siddiqui AJ, Dellgren G, et al. Angiogenic effects of sequential release of VEGF-A165 and PDGF-BB with alginate hydrogels after myocardial infarction. Cardiovasc Res 2007;75(1):178–85. [143] Madrigal JL, Sharma SN, Campbell KT, Stilhano RS, Gijsbers R, Silva EA. Microgels produced using microfluidic on-chip polymer blending for controlled released of VEGF encoding lentivectors. Acta Biomater 2018;69:265–76. [144] Kong HJ, Kim ES, Huang Y-C, Mooney DJ. Design of biodegradable hydrogel for the local and sustained delivery of angiogenic plasmid DNA. Pharm Res 2008;25(5):1230–8. [145] Han Y-F, Han Y-Q, Pan Y-G, Chen Y-L, Chai J-K. Transplantation of microencapsulated cells expressing VEGF improves angiogenesis in implanted xenogeneic acellular dermis on wound. Transplant Proc 2010;42(5):1935–43. [146] Alsberg E, Anderson KW, Albeiruti A, Franceschi RT, Mooney DJ. Cell-interactive alginate hydrogels for bone tissue engineering. J Dent Res 2001;80(11):2025–9. [147] Dhoot  NO, Tobias  CA, Fischer  I, Wheatley  MA. Peptide-modified alginate surfaces as a growth permissive substrate for neurite outgrowth. J Biomed Mater Res A 2004;71A(2):191–200. [148] Koo  LY, Irvine  DJ, Mayes  AM, Lauffenburger  DA, Griffith  LG. Co-regulation of cell adhesion by nanoscale RGD organization and mechanical stimulus. J Cell Sci 2002;115(7):1423–33. [149] Bouhadir KH, Lee KY, Alsberg E, Damm KL, Anderson KW, Mooney DJ. Degradation of partially oxidized alginate and its potential application for tissue engineering. Biotechnol Prog 2001;17(5):945–50. [150] Campbell KT, Stilhano RS, Silva EA. Enzymatically degradable alginate hydrogel systems to deliver endothelial progenitor cells for potential revasculature applications. Biomaterials 2018;179:109–21.

144

4.  Bioengineering strategies for gene delivery

[151] Medved  L, Litvinovich  S, Ugarova  T, Matsuka  Y, Ingham  K. Domain structure and functional activity of the recombinant human fibrinogen γ-module (γ148−411)†. Biochemistry 1997;36(15):4685–93. [152] Tennent GA, Brennan SO, Stangou AJ, O’Grady J, Hawkins PN, Pepys MB. Human plasma fibrinogen is synthesized in the liver. Blood 2007;109(5):1971–4. [153] Brown AC, Barker TH. Fibrin-based biomaterials: modulation of macroscopic properties through rational design at the molecular level. Acta Biomater 2014;10(4):1502–14. [154] Jennewein C, Tran N, Paulus P, Ellinghaus P, Eble JA, Zacharowski K. Novel aspects of fibrin(ogen) fragments during inflammation. Mol Med 2011;17(5):568–73. [155] Weisel  JW. Structure of fibrin: impact on clot stability. J Thromb Haemost 2007;5(s1):116–24. [156] Sánchez-Cortés J, Mrksich M. The platelet integrin αIIbβ3 binds to the RGD and AGD motifs in fibrinogen. Chem Biol 2009;16(9):990–1000. [157] Kidd ME, Shin S, Shea LD. Fibrin hydrogels for lentiviral gene delivery in vitro and in vivo. J Control Release 2012;157(1):80–5. [158] Trentin D, Hall H, Wechsler S, Hubbell JA. Peptide-matrix-mediated gene transfer of an oxygen-insensitive hypoxia-inducible factor-1 variant for local induction of angiogenesis. Proc Natl Acad Sci U S A 2006;103(8):2506–11. [159] Thiersch M, Rimann M, Panagiotopoulou V, Öztürk E, Biedermann T, Textor M, et al. The angiogenic response to PLL-g-PEG-mediated HIF-1α plasmid DNA delivery in healthy and diabetic rats. Biomaterials 2013;34(16):4173–82. [160] Friess  W. Collagen—biomaterial for drug delivery 1 dedicated to Professor Dr. Eberhard Nürnberg, Friedrich-Alexander-Universität Erlangen-Nürnberg, on the occasion of his 70th birthday.1. Eur J Pharm Biopharm 1998;45(2):113–36. [161] Elzoghby AO, Samy WM, Elgindy NA. Protein-based nanocarriers as promising drug and gene delivery systems. J Control Release 2012;161(1):38–49. [162] Wang  H, Boerman  OC, Sariibrahimoglu  K, Li  Y, Jansen  JA, Leeuwenburgh  SCG. Comparison of micro- vs. nanostructured colloidal gelatin gels for sustained delivery of osteogenic proteins: bone morphogenetic protein-2 and alkaline phosphatase. Biomaterials 2012;33(33):8695–703. [163] Bode  F, da Silva  MA, Drake  AF, Ross-Murphy  SB, Dreiss  CA. Enzymatically cross-linked tilapia gelatin hydrogels: physical, chemical, and hybrid networks. Biomacromolecules 2011;12(10):3741–52. [164] Zou  D, Zhang  Z, He  J, Zhang  K, Ye  D, Han  W, et  al. Blood vessel formation in the ­tissue-engineered bone with the constitutively active form of HIF-1α mediated BMSCs. Biomaterials 2012;33(7):2097–108. [165] Ciucurel  EC, Sefton  MV. Del-1 overexpression in endothelial cells increases vascular density in tissue-engineered implants containing endothelial cells and adipose-­ derived mesenchymal stromal cells. Tissue Eng A 2014;20(7–8):1235–52. [166] Liu G, Wang X, Sun X, Deng C, Atala A, Zhang Y. The effect of urine-derived stem cells expressing VEGF loaded in collagen hydrogels on myogenesis and innervation following after subcutaneous implantation in nude mice. Biomaterials 2013;34(34):8617–29. [167] Kasahara H, Tanaka E, Fukuyama N, Sato E, Sakamoto H, Tabata Y, et al. Biodegradable gelatin hydrogel potentiates the angiogenic effect of fibroblast growth factor 4 plasmid in rabbit hindlimb ischemia. J Am Coll Cardiol 2003;41(6):1056–62. [168] Mao Z, Shi H, Guo R, Ma L, Gao C, Han C, et al. Enhanced angiogenesis of porous collagen scaffolds by incorporation of TMC/DNA complexes encoding vascular endothelial growth factor. Acta Biomater 2009;5(8):2983–94. [169] Ylä-Herttuala S, Bridges C, Katz MG, Korpisalo P. Angiogenic gene therapy in cardiovascular diseases: dream or vision? Eur Heart J 2017;38(18):1365–71. [170] Mozaffarian D, Benjamin EJ, Go AS, Arnett DK, Blaha MJ, Cushman M, et al. Heart disease and stroke statistics—2016 update: a report from the American Heart Association. Circulation 2016;133(4):38–48.



References

145

[171] Townsend  N, Nichols  M, Scarborough  P, Rayner  M. Cardiovascular disease in Europe—epidemiological update 2015. Eur Heart J 2015;36(40):2696–705. [172] Cerbone  AM, Macarone-Palmieri  N, Saldalamacchia  G, Coppola  A, Di Minno  G, Rivellese AA. Diabetes, vascular complications and antiplatelet therapy: open problems. Acta Diabetol 2009;46(4):253–61. [173] Hirsch AT, Criqui MH, Treat-Jacobson D, Regensteiner JG, Creager MA, Olin JW, et al. Peripheral arterial disease detection, awareness, and treatment in primary care. JAMA 2001;286(11):1317–24. [174] Go  AS, Mozaffarian  D, Roger Véronique  L, Benjamin Emelia  J, Berry  JD, Blaha Michael J, et al. Executive summary: heart disease and stroke statistics—2014 update. Circulation 2014;129(3):399–410. [175] Simmons A, Steffen K, Sanders S. Medical therapy for peripheral arterial disease. Curr Opin Cardiol 2012;27(6):592–7. [176] Jackson EA, Munir K, Schreiber T, Rubin JR, Cuff R, Gallagher KA, et al. Impact of sex on morbidity and mortality rates after lower extremity interventions for peripheral arterial disease. J Am Coll Cardiol 2014;63(23):2525–30. [177] Silva EA, Mooney DJ. Effects of VEGF temporal and spatial presentation on angiogenesis. Biomaterials 2010;31(6):1235. [178] Silva EA, Kim E-S, Kong HJ, Mooney DJ. Material-based deployment enhances efficacy of endothelial progenitor cells. Proc Natl Acad Sci U S A 2008;105(38):14347–52. [179] Laschke MW, Menger MD. Vascularization in tissue engineering: angiogenesis versus inosculation. Eur Surg Res 2012;48(2):85–92. [180] Tsang  VL, Bhatia  SN. Fabrication of three-dimensional tissues. In: Lee  K, Kaplan  D, editors. Tissue engineering II: Basics of tissue engineering and tissue applications. Advances in biochemical engineering/biotechnologyBerlin/Heidelberg: Springer Berlin Heidelberg; 2007. p. 189–205. https://doi.org/10.1007/10_010. [cited 2018 Dec 22]. [181] Chen W, Tabata Y, Wah Tong Y. Fabricating tissue engineering scaffolds for simultaneous cell growth and drug delivery. Curr Pharm Des 2010;16(21):2388–94. [182] Bearzi  C, Gargioli  C, Baci  D, Fortunato  O, Shapira-Schweitzer  K, Kossover  O, et  al. PlGF–MMP9-engineered iPS cells supported on a PEG–fibrinogen hydrogel scaffold possess an enhanced capacity to repair damaged myocardium. Cell Death Dis 2014;5(2):e1053. [183] Liu  Z, Chang  H, Hou  Y, Wang  Y, Zhou  Z, Wang  M, et  al. Lentivirus-mediated ­microRNA-26a overexpression in bone mesenchymal stem cells facilitates bone regeneration in bone defects of calvaria in mice. Mol Med Rep 2018;18(6):5317–26. [184] Liu T, Sheng W, Chen Y, Xie Y, Miao J, Yang H, et al. Study on inducing angiogenesis of regenerated silk fibroin film modified by the Ad-Ang-1 transgenic fibroblasts. In: 2010 4th international conference on bioinformatics and biomedical engineering; 2010. p. 1–6. [185] Baumann L, Prokoph S, Gabriel C, Freudenberg U, Werner C, Beck-Sickinger AG. A novel, biased-like SDF-1 derivative acts synergistically with starPEG-based heparin hydrogels and improves eEPC migration in vitro. J Control Release 2012;162(1):68–75. [186] Browne S, Monaghan MG, Brauchle E, Berrio DC, Chantepie S, Papy-Garcia D, et al. Modulation of inflammation and angiogenesis and changes in ECM GAG-activity via dual delivery of nucleic acids. Biomaterials 2015;69:133–47. [187] Zhou W, He D-Q, Liu J-Y, Feng Y, Zhang X-Y, Hua C-G, et al. Angiogenic gene-­modified myoblasts promote vascularization during repair of skeletal muscle defects. J Tissue Eng Regen Med 2015;9(12):1404–16. [188] Dash BC, Thomas D, Monaghan M, Carroll O, Chen X, Woodhouse K, et al. An injectable elastin-based gene delivery platform for dose-dependent modulation of angiogenesis and inflammation for critical limb ischemia. Biomaterials 2015;65:126–39. [189] Tokatlian T, Cam C, Segura T. Non-viral DNA delivery from porous hyaluronic acid hydrogels in mice. Biomaterials 2014;35(2):825–35.

146

4.  Bioengineering strategies for gene delivery

[190] Paquet J, Moya A, Bensidhoum M, Petite H. Engineered cell-free scaffold with twostage delivery of miRNA-26a for bone repair. Ann Transl Med 2016;4(10). [cited 2018 Dec 23]. Available from, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4885895/. [191] Lee PC, Salyapongse AN, Bragdon GA, Shears LL, Watkins SC, Edington HDJ, et al. Impaired wound healing and angiogenesis in eNOS-deficient mice. Am J Physiol Heart Circ Physiol 1999;277(4):H1600–8. [192] Norton  WT, Aquino  DA, Hozumi  I, Chiu  F-C, Brosnan  CF. Quantitative aspects of reactive gliosis: a review. Neurochem Res 1992;17(9):877–85. [193] Seeley WW. Mapping neurodegenerative disease onset and progression. Cold Spring Harb Perspect Biol 2017;9(8):a023622. [194] Taylor JP, Hardy J, Fischbeck KH. Toxic proteins in neurodegenerative disease. Science 2002;296(5575):1991–5. [195] Aguado C, Sarkar S, Korolchuk VI, Criado O, Vernia S, Boya P, et al. Laforin, the most common protein mutated in Lafora disease, regulates autophagy. Hum Mol Genet 2010;19(14):2867–76. [196] Smith  RA, Miller  TM, Yamanaka  K, Monia  BP, Condon  TP, Hung  G, et  al. Antisense oligonucleotide therapy for neurodegenerative disease. J Clin Invest 2006;116(8):2290–6. [197] Ross CA, Poirier MA. Protein aggregation and neurodegenerative disease. Nat Med 2004;10(7s):S10–7. [198] Morgan D, Diamond DM, Gottschall PE, Ugen KE, Dickey C, Hardy J, et al. Aβ peptide vaccination prevents memory loss in an animal model of Alzheimer’s disease. Nature 2000;408(6815):982–5. [199] Orive G, Anitua E, Pedraz JL, Emerich DF. Biomaterials for promoting brain protection, repair and regeneration. Nat Rev Neurosci 2009;10(9):682–92. [200] Pardridge  WM. The blood-brain barrier: bottleneck in brain drug development. NeuroRX 2005;2(1):3–14. [201] Xia H, Mao Q, Paulson HL, Davidson BL. siRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol 2002;20(10):1006–10. [202] Xia  H, Mao  Q, Eliason  SL, Harper  SQ, Martins  IH, Orr  HT, et  al. RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia. Nat Med 2004;10(8):816–20. [203] Singer  O, Marr  RA, Rockenstein  E, Crews  L, Coufal  NG, Gage  FH, et  al. Targeting BACE1 with siRNAs ameliorates Alzheimer disease neuropathology in a transgenic model. Nat Neurosci 2005;8(10):1343–9. [204] Raoul C, Abbas-Terki T, Bensadoun J-C, Guillot S, Haase G, Szulc J, et al. Lentiviralmediated silencing of SOD1 through RNA interference retards disease onset and progression in a mouse model of ALS. Nat Med 2005;11(4):423–8. [205] Mazarakis ND, Azzouz M, Rohll JB, Ellard FM, Wilkes FJ, Olsen AL, et al. Rabies virus glycoprotein pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral delivery. Hum Mol Genet 2001;10(19):2109–21. [206] Azzouz M, Ralph GS, Storkebaum E, Walmsley LE, Mitrophanous KA, Kingsman SM, et al. VEGF delivery with retrogradely transported lentivector prolongs survival in a mouse ALS model. Nature 2004;429(6990):413–7. [207] Kaspar BK, Lladó J, Sherkat N, Rothstein JD, Gage FH. Retrograde viral delivery of IGF-1 prolongs survival in a mouse ALS model. Science 2003;301(5634):839–42. [208] Freudenberg U, Hermann A, Welzel PB, Stirl K, Schwarz SC, Grimmer M, et al. A starPEG–heparin hydrogel platform to aid cell replacement therapies for neurodegenerative diseases. Biomaterials 2009;30(28):5049–60. [209] Saraiva  C, Praça  C, Ferreira  R, Santos  T, Ferreira  L, Bernardino  L. Nanoparticlemediated brain drug delivery: overcoming blood–brain barrier to treat neurodegenerative diseases. J Control Release 2016;235:34–47.



References

147

[210] Begley DJ. Delivery of therapeutic agents to the central nervous system: the problems and the possibilities. Pharmacol Ther 2004;104(1):29–45. [211] Kreuter J, Ramge P, Petrov V, Hamm S, Gelperina SE, Engelhardt B, et al. Direct evidence that polysorbate-80-coated poly(butylcyanoacrylate) nanoparticles deliver drugs to the CNS via specific mechanisms requiring prior binding of drug to the nanoparticles. Pharm Res 2003;20(3):409–16. [212] Kanter-Schlifke  I, Fjord-Larsen  L, Kusk  P, Ängehagen  M, Wahlberg  L, Kokaia  M. GDNF released from encapsulated cells suppresses seizure activity in the epileptic hippocampus. Exp Neurol 2009;216(2):413–9. [213] Sajadi  A, Bensadoun  J-C, Schneider  BL, Lo Bianco  C, Aebischer  P. Transient striatal delivery of GDNF via encapsulated cells leads to sustained behavioral improvement in a bilateral model of Parkinson disease. Neurobiol Dis 2006;22(1):119–29. [214] Spuch C, Antequera D, Portero A, Orive G, Hernández RM, Molina JA, et al. The effect of encapsulated VEGF-secreting cells on brain amyloid load and behavioral impairment in a mouse model of Alzheimer’s disease. Biomaterials 2010;31(21):5608–18. [215] Ross CJD, Ralph M, Chang PL. Somatic gene therapy for a neurodegenerative disease using microencapsulated recombinant cells. Exp Neurol 2000;166(2):276–86. [216] Kordower JH, Chen E-Y, Mufson EJ, Winn SR, Emerich DF. Intrastriatal implants of polymer encapsulated cells genetically modified to secrete human nerve growth f­ actor: trophic effects upon cholinergic and noncholinergic striatal neurons. Neuroscience 1996;72(1):63–77. [217] Emerich DF, Winn SR, Harper J, Hammang JP, Baetge EE, Kordower JH. Implants of polymer-encapsulated human NGF-secreting cells in the nonhuman primate: rescue and sprouting of degenerating cholinergic basal forebrain neurons. J Comp Neurol 1994;349(1):148–64. [218] Winn  SR, Hammang  JP, Emerich  DF, Lee  A, Palmiter  RD, Baetge  EE. Polymerencapsulated cells genetically modified to secrete human nerve growth factor promote the survival of axotomized septal cholinergic neurons. Proc Natl Acad Sci U S A 1994;91(6):2324–8. [219] Kordower JH, Winn SR, Liu YT, Mufson EJ, Sladek JR, Hammang JP, et al. The aged monkey basal forebrain: rescue and sprouting of axotomized basal forebrain neurons after grafts of encapsulated cells secreting human nerve growth factor. Proc Natl Acad Sci 1994;91(23):10898–902. [220] Emerich DF, Hammang JP, Baetge EE, Winn SR. Implantation of polymer-­encapsulated human nerve growth factor-secreting fibroblasts attenuates the behavioral and neuropathological consequences of quinolinic acid injections into rodent striatum. Exp Neurol 1994;130(1):141–50. [221] Emerich  DF, Lindner  MD, Winn  SR, Chen  E-Y, Frydel  BR, Kordower  JH. Implants of encapsulated human CNTF-producing fibroblasts prevent behavioral deficits and striatal degeneration in a rodent model of huntington’s disease. J Neurosci 1996;16(16):5168–81. [222] Emerich DF, Winn SR, Hantraye PM, Peschanski M, Chen E-Y, Chu Y, et al. Protective effect of encapsulated cells producing neurotrophic factor CNTF in a monkey model of Huntington’s disease. Nature 1997;386(6623):395–9. [223] Hoffman  D, Breakefield  XO, Short  MP, Aebischer  P. Transplantation of a polymer-­ encapsulated cell line genetically engineered to release NGF. Exp Neurol 1993;122(1):100–6. [224] Date I, Shingo T, Yoshida H, Fujiwara K, Kobayashi K, Takeuchi A, et al. Grafting of encapsulated genetically modified cells secreting GDNF into the striatum of parkinsonian model rats. Cell Transplant 2001;10(4):419–22. [225] Yasuhara  T, Shingo  T, Kobayashi  K, Takeuchi  A, Yano  A, Muraoka  K, et  al. Neuroprotective effects of vascular endothelial growth factor (VEGF) upon dopaminergic neurons in a rat model of Parkinson’s disease. Eur J Neurosci 2004;19(6):1494–504.

148

4.  Bioengineering strategies for gene delivery

[226] Yasuhara T, Shingo T, Muraoka K, Kobayashi K, Takeuchi A, Yano A, et al. Early transplantation of an encapsulated glial cell line—derived neurotrophic factor—producing cell demonstrating strong neuroprotective effects in a rat model of Parkinson disease. J Neurosurg 2005;102(1):80–9. [227] Yasuhara T, Shingo T, Muraoka K, Kameda M, Agari T, Ji YW, et al. Neurorescue effects of VEGF on a rat model of Parkinson’s disease. Brain Res 2005;1053(1):10–8. [228] Bachoud-Lévi  A-C, Déglon  N, Nguyen  J-P, Bloch  J, Bourdet  C, Winkel  L, et  al. Neuroprotective gene therapy for huntington’s disease using a polymer encapsulated BHK cell line engineered to secrete human CNTF. Hum Gene Ther 2000;11(12):1723–9. [229] Mittoux  V, Joseph  J-M, Conde  F, Palfi  S, Dautry  C, Poyot  T, et  al. Restoration of cognitive and motor functions by ciliary neurotrophic factor in a primate model of Huntington’s disease. Hum Gene Ther 2000;11(8):1177–88. [230] Bloch  J, Bachoud-Lévi  AC, Déglon  N, Lefaucheur  JP, Winkel  L, Palfi  S, et  al. Neuroprotective gene therapy for Huntington’s disease, using polymer-encapsulated cells engineered to secrete human ciliary neurotrophic factor: results of a phase I study. Hum Gene Ther 2004;15(10):968–75. [231] Pochon N-M, Heyd B, Déglon N, Joseph J-M, Zurn AD, Baetge EE, et al. Gene therapy for amyotrophic lateral sclerosis (ALS) using a polymer encapsulated xenogenic cell line engineered to secrete hCNTF. Lausanne University Medical School, Lausanne, Switzerland. Hum Gene Ther 1996;7(7):851–60. [232] Aebischer P, Schluep M, Déglon N, Joseph J-M, Hirt L, Heyd B, et al. Intrathecal delivery of CNTF using encapsulated genetically modifiedxenogeneic cells in amyotrophic lateral sclerosis patients. Nat Med 1996;2(6):696–9. [233] Wahlberg  LU, Lind  G, Almqvist  PM, Kusk  P, Tornøe  J, Juliusson  B, et  al. Targeted delivery of nerve growth factor via encapsulated cell biodelivery in Alzheimer disease: a technology platform for restorative neurosurgery: clinical article. J Neurosurg 2012;117(2):340–7. [234] Eriksdotter-Jönhagen M, Linderoth B, Lind G, Aladellie L, Almkvist O, Andreasen N, et  al. Encapsulated cell biodelivery of nerve growth factor to the basal forebrain in patients with Alzheimer’s disease. Dement Geriatr Cogn Disord 2012;33(1):18–28. [235] Henderson CE, Phillips HS, Pollock RA, Davies AM, Lemeulle C, Armanini M, et al. GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle. Science 1994;266(5187):1062–4. [236] Choi-Lundberg  DL, Lin  Q, Chang  Y-N, Chiang  YL, Hay  CM, Mohajeri  H, et  al. Dopaminergic neurons protected from degeneration by GDNF gene therapy. Science 1997;275(5301):838–41. [237] Zurn AD, Henry H, Schluep M, Aubert V, Winkel L, Eilers B, et al. Evaluation of an intrathecal immune response in amyotrophic lateral sclerosis patients implanted with encapsulated genetically engineered xenogeneic cells. Cell Transplant 2000;9(4):471–84. [238] Gorelick  PB. Risk factors for vascular dementia and Alzheimer disease. Stroke 2004;35(11 Suppl. 1):2620–2. [239] Casserly I, Topol EJ. Convergence of atherosclerosis and Alzheimer’s disease: inflammation, cholesterol, and misfolded proteins. The Lancet 2004;363(9415):1139–46. [240] Farkas  E, Luiten  PGM. Cerebral microvascular pathology in aging and Alzheimer’s disease. Prog Neurobiol 2001;64(6):575–611.