Gene therapy for the CNS using AAVs: The impact of systemic delivery by AAV9

    Gene therapy for the CNS using AAVs: The impact of systemic delivery by AAV9 Joana Saraiva, Rui Jorge Nobre, Luis Pereira de Almeida PII: DOI: Reference:

S0168-3659(16)30736-2 doi: 10.1016/j.jconrel.2016.09.011 COREL 8465

To appear in:

Journal of Controlled Release

Received date: Revised date: Accepted date:

21 May 2016 9 September 2016 12 September 2016

Please cite this article as: Joana Saraiva, Rui Jorge Nobre, Luis Pereira de Almeida, Gene therapy for the CNS using AAVs: The impact of systemic delivery by AAV9, Journal of Controlled Release (2016), doi: 10.1016/j.jconrel.2016.09.011

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ACCEPTED MANUSCRIPT Gene therapy for the CNS using AAVs: the impact of systemic delivery by AAV9 Joana Saraiva1*, Rui Jorge Nobre1, 2*, Luis Pereira de Almeida1, 3 1

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CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; Institute for Interdisciplinary Research, University of Coimbra, Portugal; 3Faculty of Pharmacy, University of Coimbra, Portugal. *Equal contribution

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Corresponding author: Luís Pereira de Almeida CNC - Center for Neuroscience and Cell Biology University of Coimbra Rua Larga 3004-504 Coimbra, Portugal. Email: [email protected]

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Keywords: Central Nervous System, Gene therapy, Adeno-associated Virus (AAV), AAV9, systemic administration

ACCEPTED MANUSCRIPT Gene therapy for the CNS using AAVs: the impact of systemic delivery by AAV9

Abstract

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Several attempts have been made to discover the ideal vector for gene therapy in central nervous system (CNS). Adeno-associated viruses (AAVs) are currently the

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preferred vehicle since they exhibit stable transgene expression in post-mitotic cells, neuronal tropism, low risk of insertional mutagenesis and diminished immune responses. Additionally, the discovery that a particular serotype, AAV9, bypasses the

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blood-brain barrier has raised the possibility of intravascular administration as a noninvasive delivery route to achieve widespread CNS gene expression. AAV9 intravenous

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delivery has already shown promising results for several diseases in animal models, including lysosomal storage disorders and motor neuron diseases, opening the way to the first clinical trial in the field. This review presents an overview of clinical trials for

Introduction

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gene delivery using AAV9.

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CNS disorders using AAVs and will focus on preclinical studies based on the systemic

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Gene therapy uses nucleic acids as therapeutic agents to permanently correct a disease. This manipulation can be achieved using different strategies, such as: i) introducing a functional copy of a defective gene; ii) silencing a mutant allele using RNA interference (RNAi) systems; iii) introducing a disease-modifying gene; or iv) using gene-editing methods. This approach is promising for many central nervous system (CNS) disorders, usually divided into two categories: i) Monogenic diseases, caused by defects in single genes, such as lysosomal storage disorders (LSDs) or polyglutamine diseases and ii) Multifactorial disorders, in which neuronal populations are lost or dysfunctional due to genetic predispositions and/or environmental factors [1]. In both cases, gene therapy is a potential alternative to traditional pharmacologic approaches, which do not provide full recovery and present considerable side effects. In addition, this strategy is able to directly and permanently correct the genetic defects, avoiding repeated treatments [2]. 1

ACCEPTED MANUSCRIPT The success of CNS gene therapy approaches greatly depends on the selected delivery system. Non-viral vectors (e.g. liposomes, exosomes, polymeric nanoparticles) are considered a promising option, due to their simple and cost-effective production

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methods, as well as their safety profile [3, 4]. However, they present a relatively low

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efficiency and mediate a transient effect, requiring repeated administrations with the

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potential risk of triggering an immune response. Therefore, recombinant viral vectors are considered the most efficient system to achieve long-term and stable gene expression in the CNS. Over the years, several viral vector systems have been investigated for this purpose, such as: herpes simplex virus type 1, adenovirus, adeno-

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associated virus (AAV) and lentivirus (LV) (reviewed in [5]).

AAV and lentiviral vectors have emerged as the vectors of choice for CNS gene transfer

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[6]. Although both exhibit a limited packaging capacity when compared to adenoviruses and herpes simplex viruses, they present significant advantages,

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including long-lasting gene expression and no toxicity. Accordingly, extensive pre-

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clinical studies have successfully shown the therapeutic potential of both vectors in neurological disorders [7-12]. When comparing these two options, AAVs exhibit

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important advantages, including a better safety profile due to the non-pathogenic nature of their wild-type form. Moreover, AAVs normally induce higher transgene expression levels than LVs, as well as a larger vector spread (1-3 mm versus 500-700

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μm) after direct infusion in the brain [13]. Taking everything into account, AAVs mediate a safe, widespread and robust transgene expression, thereby being the most common vectors in clinical studies for CNS disorders. In the present review we will summarize the main properties of AAVs as gene delivery vectors, emphasizing the potential of a particular serotype, the AAV9, and the most relevant pre-clinical and clinical studies using this serotype.

1. Adeno-associated virus (AAV): the basics

AAV is a nonenveloped, single-stranded DNA-containing virus, firstly defined as a contaminant of adenovirus preps. This virus belongs to the Parvoviridae family, particularly the genus Dependovirus, since it needs co-infection with a helper virus to replicate and complete its life cycle [14]. Its 4700 bp genome comprises two open 2

ACCEPTED MANUSCRIPT reading frames (ORFs), rep and cap, flanked by inverted terminal repeats (ITRs) on the 5’ and 3’ ends, as illustrated in Figure 1. The rep ORF encodes four proteins: Rep 40, 52, 68 and 78, corresponding to spliced and unspliced products expressed using

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different promoters (P5 and P19). These Rep proteins, named based on their molecular

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weight, are needed for AAV replication, transcription, integration and encapsidation.

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On the other hand, the cap ORF leads to the production of three structural proteins (VP1, VP2 and VP3), which assemble in a ratio of 1:1:10 to produce an icosahedral capsid of approximately 25 nm in diameter. The cap ORF is transcribed from the P40 promoter and generates these three transcripts through alternative splicing and

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different start codons [15]. Lastly, an alternate ORF in cap encodes assembly-activating

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protein (AAP), which cooperate with VP1-3, contributing to capsid assembly [16].

Figure 1 – Representation of the wild-type adeno-associated virus genome (wt-AAV), containing two inverted terminal repeats (ITRs), three promoters (P5, P19 and P40), two ORFs (rep and cap), and a polyadenylation site (pA). Rep proteins are generated from the spliced and unspliced transcripts controlled by the p5 promoter (Rep68 and Rep78) and p19 promoter (Rep40 and Rep52). The P40 promoter initiates cap gene transcription. The unspliced transcript originates VP1; whereas the spliced variants encode VP2 from an alternative start codon (ACG) and VP3 from the conventional start codon. An alternate ORF encodes assembly-activating protein (AAP). Over the last years, twelve natural serotypes and more than one hundred variants of AAVs have been detected and isolated from humans and other primates 3

ACCEPTED MANUSCRIPT [17]. Different serotypes are defined by capsid protein motifs that are identified by distinct neutralizing antibodies. Besides antigenicity, AAV serotypes present characteristic properties concerning capsid-receptor interactions. In fact, certain

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exposed capsid regions define interactions with the principal AAV receptors, which are

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usually cell surface glycans. For instance, AAV2 binds heparin sulfate proteoglycans

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and AAV9 requires N-terminal galactose residues [18]. These differences in receptoraffinity patterns are important in determining the preferential tissue tropism of AAV serotypes. For example, AAV 8 and 9 are considered the optimal serotypes to target the liver, while AAV 5 and 9 are the most suitable for lung transduction. The

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corresponding species origin, known receptors, homology with AAV2, natural tropism,

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as well as other properties of each serotype are indicated in Table 1.

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Table 1 – Principal characteristics of AAV serotypes (AAV 1-9). Natural serotypes

Recombinant AAV vectors (rAAVs)

Serotype

Origin Primary Receptor

NHP

α2,3/α2,6 N-linked ? SA

AAV2

Human

HSPG

AAV3

NHP

HSPG

AAV4

NHP

α2,3 O-linked SA

Human

α2,3 N-linked SA

Human

α2,3/α2,6 N-linked SA/ HSPG

AAV5

AAV6

Coreceptor

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AAV1

Capsid homology to AAV2 (%)

NAbs in humans (%)

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AAV receptors

HGFR LamR FGFR-1 Integrin

83

67

100

72

FGFR-1 integrin HGFR LamR

88

?

?

60

?

PDGFR

EGFR

57

83

40

46

Tissue tropism a)

SM, CNS, heart, lung, eye, pancreas Kidney, SM, CNS, liver, eye

SM, HCC

Eye, CNS

CNS, lung, eye, SM SM (IV), heart, lung

CNS transduction Overall level of transduction b)

Kinetics b)

Moderate

Upon intraparenchymal injection in adult mice

Upon IV injection in neonatal mice

Level of transduction

Cellular tropism

Level of transduction

Cellular Tropism

Rapid

++

Neurons + glia

+

Neurons + glia

R

Low

Slow

+

Neurons

-

-

A

Low

Slow

?

?

?

?

?

Low

Slow

+

Ependymal cells

?

?

?

Low

Slow

+++

Neurons + glia

-

-

R

Moderate

Rapid

++

?

+

Neurons + glia

R

4

Axonal transport

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Human

N-linked Galactose

LamR

83

82

38

47

SM, eye, CNS, liver

Liver, SM, CNS, eye, pancreas, heart Liver, lung, SM (IV), heart(IV), CNS (IV), pancreas, eye, kidney (IV) ,

High

Rapid

+++

Neurons

+

Neurons + glia

?

Moderate

Rapid

+++

Neurons

+

Neurons + glia

A, R

++

Neurons + glia

A, R

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LamR

?

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?

82

High

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NHP

?

Rapid

+++

Neurons

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a) Preferential tropism in mammals after local delivery. The tissues for which each serotype is considered the preferential one are written in bold. When “IV” is indicated, the tropism is also observed upon intravenous injection. b) The results were obtained by bioluminescence imaging following AAV systemic injection in male adult mice. Adapted from: [1, 15],[19, 20]

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Abbreviations: NHP - Non-human primates; ? – Unknown; HGFR - hepatocyte growth factor receptor; LamR – Laminin Receptor; FGFR1 - fibroblast growth factor receptor 1; HSPG - Heparan sulfate proteoglycan; EGFR - epidermal growth factor receptor; PDGFR - platelet-derived growth factor receptor; NAbs – neutralizing antibodies; SM- skeletal muscle; HCC- hepatocellular carcinoma; IV- intravenous; - : no transduction; + : low levels; ++ : moderate levels; +++ : high levels; A : anterograde transport; R : Retrograde transport.

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AAV9

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AAV8

NHP

Successful transduction by AAV vectors starts with cell surface receptor binding

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AAV7

and depends on several subsequent steps, such as endocytic uptake, escape from the endosomal pathway, entry into the cell nucleus, virus uncoating and single-stranded genome release, second-strand synthesis and finally transcription [1, 15]. After cell infection, the virus might follow two different life cycle pathways. If a helper virus is available (e.g. adenovirus or herpesvirus), AAV engages in a lytic stage, which involves rapid replication within the cell, releasing new particles into the environment. However, in the lack of helper viruses, AAV enters in a latent stage, in which the genome can either persist in an extrachromosomal state or integrate into the host cell genome [15]. This integration event happens in a specific site in the q arm of human chromosome 19 (AAVS1), in a process that depends on AAV ITRs and Rep proteins [21]. In fact, Rep68 or Rep78 bind to RBEs (Rep binding elements) within AAV-ITRs and AAVS1, consequently forming a complex [22] and enabling integration into AAVS1 [23]. 5

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2. Recombinant AAVs (rAAVs) for CNS gene therapy

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2.1 Production of rAAVs

The relative simplicity of the wild-type AAV genome facilitates the design of

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recombinant-AAVs (rAAVs) as gene therapy vectors. The process of rAAV production generally involves transient triple transfection of HEK0293 cells (illustrated in Figure 2) with the following plasmids: 1) the transfer vector, containing an ITR-flanked

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expression cassette that comprises a promoter, the transgene and a poly-adenylation (PolyA) signal or a string of five thymidines to terminate transcription. Replication and

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packaging of the vector genome during production is regulated by the ITR elements

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alone and thus no AAV coding regions are present in these recombinant vectors [24]; 2) the helper plasmid (pHelper), containing adenoviral regions E1, E2, E3 and VA,

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which contribute to genome replication [25]; 3) the pAAV-RC, a plasmid containing rep and cap genes that mediate genome replication and capsid assembly respectively. In this process, the genome is packaged into an AAV capsid, leading to a rAAV vector with

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a total packaging capacity of approximately 5 kb [26], which is subsequently purified and used in gene therapy. Importantly, since rAAVs do not carry any viral coding sequence, target cells will not produce any viral product.

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Figure 2 – Representation of recombinant AAV (rAAV) generation process, which involves transient triple transfection of HEK0293 cells using three plasmids: 1) the transfer vector, 2) the helper plasmid (pHelper), and 3) the pAAV-RC plasmid. After replication and capsid assembly, the genome is packaged into an AAV capsid, resulting in new virus formation and releasing from transfected cells. Abbreviations: pA - poly(A) tail; ITR – inverted terminal repeat.

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2.2 – Principal properties of rAAVs

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AAV2 was the first serotype to be modified into a recombinant vector for gene delivery [27]. Recently, rAAVs originated from all 12 natural serotypes have been generated, showing different properties (see Table 1). Zincarelli and collaborators, for

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instance, examined transgene expression levels and AAV biodistribution after intravenous injection in adult male mice. Based on bioluminescence imaging data, they grouped these serotypes into different categories regarding the levels of transduction (as low, moderate or high) and kinetics (rapid or slow-onset) [28]. The ability of rAAVs to target the CNS has been studied using different delivery routes. When administrated into the brain parenchyma of rodents, the serotypes exhibit different CNS transduction efficiency and cellular tropism. Several comparative analyses have revealed that most of the serotypes are superior to AAV2, as they transduce larger CNS areas and present higher transgene expression levels upon intraparenchymal injection, such as AAV5, AAV7, AAV8 [29, 30] and particularly AAV9, which shows the highest vector distribution throughout the CNS [31]. Furthermore, the great majority of rAAV serotypes transduce almost exclusively neurons [31]. AAV4, on 7

ACCEPTED MANUSCRIPT the contrary, preferentially targets ependymal cells, whereas AAV1 and AAV5 efficiently transduce both neurons and glial cells [32] [33]. One important pathway that might facilitate vector spread within the CNS is axonal transport, following a

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retrograde and/or anterograde direction. In this way, viral vectors are transported

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across synaptic connections, ultimately transducing spatially different neuronal

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subpopulations. AAV9, for instance, undergoes both anterograde and retrograde transport, which might contribute to its wide distribution throughout the CNS [34]. Finally, Zhang and Yang et al. [35, 36] evaluated CNS transduction after intravenous injection in neonatal and adult mice respectively. Based on the results

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obtained in newborn animals, the different serotypes can be ranked according to their performance in the following order: 1) AAV9, which displays the greatest CNS

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transduction efficiency; 2) AAV 1, 6, 7, 8 which show moderate transduction levels and 3) AAV2 and 5, for which the authors only observed small populations of transduced

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cells. Following this type of administration, AAV serotypes have the capacity to

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transduce both neurons and astrocytes [35, 37]. To summarize, Table 1 provides a direct comparison between the most

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commonly used rAAVs, regarding tissue tropism, overall expression levels and kinetics, as well as their ability to target the CNS.

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2.3. General advantages and limitations

Ideally, vectors for CNS gene delivery should present: i) an effective transduction and no off-target effects; ii) suitable transgene expression levels and duration, in order to induce a therapeutic effect in the absence of cellular toxicity; iii) lack of pathogenicity and immunogenicity, leading to no adverse responses to the treatment; and iv) large-scale efficient vector production, with high purity levels [38]. Many AAV properties fit these requirements, explaining why this vector system is currently the most used in CNS preclinical and clinical studies. In fact, AAVs transduce both mitotic and post-mitotic cells. Additionally, most AAV serotypes present neuronal tropism, as indicated in Table 1, while some are also able to transduce other CNS cell types, such as astrocytes [32]. Another advantage of this system is the stable transgene expression in CNS for the lifespan of mice [37], at least 8 8

ACCEPTED MANUSCRIPT years in non-human primates [39] and 10 years in the human brain [40]. This persistent gene expression in non-dividing cells explains why rAAVs are so suitable for gene therapy of CNS disorders, which are mostly chronic and affect post-mitotic cells.

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In summary, AAV vectors offer the opportunity of permanently correcting a

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disease through a single administration. Furthermore, no significant adverse effects

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have been observed, since AAVs are non-pathogenic, leading to diminished inflammatory and immune responses [41, 42]. In addition, rAAV cell infection results mainly in episomal transgene expression, consequently reducing the risk of insertional mutagenesis [43], an important safety concern for integrating viral vectors such as

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lentiviruses. Finally, efficient and scalable methods for rAAV production and purification provide encouragement for future investigation and clinical applications

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[44].

However, AAVs also present some limitations, stressing the importance of

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further optimization methods. One of these disadvantages concerns the delay in

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transgene expression (around 2 weeks for maximum expression) when compared to other vectors, since it needs a second-strand synthesis [45]. Self-complementary AAV

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vectors (scAAV) overcome this limitation as they carry double-stranded DNA genomes that become transcriptionally active immediately upon decapsidation in the nucleus. These scAAV vectors carry a mutated ITR, missing the terminal resolution (TR) site

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where Rep proteins cut the genomes during replication to generate ssDNA. In the absence of the TR site in one ITR, the replication continues in the opposite direction without resolution of the previous strand resulting in the synthesis of a doublestranded DNA vector genome. The double-stranded nature of the scAAV vector also means that transgene capacity is reduced to about 2.2kb [46], which is roughly half of single-stranded AAV vectors. The smaller transgene capacity imposes further limitations on promoter sizes and entities that can be expressed from these vectors. Nevertheless, it is still possible to achieve remarkable therapeutic effects within the 2.2kb constraint, by selecting small transgenes, potent small promoters and RNA interference molecules, for example. Extensive investigation of AAVs has led to other important discoveries and consequent advances in the field. One of such advances involves gene silencing, one of the main therapeutic approaches, through recruitment of the cellular RNA interference 9

ACCEPTED MANUSCRIPT (RNAi) pathway. However, some studies have recently reported cellular toxicity caused by short-hairpin RNAs (shRNAs) in mouse and rat brains [47, 48]. In fact, their high expression levels possibly induce the saturation of endogenous RNAi machinery,

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namely the exportin-5 pathway [49]. As an alternative, artificial microRNAs (miRNAs)

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are expressed at lower levels and efficiently processed, leading to no evidence of toxic

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effects. Consequently, miRNA-based platforms are nowadays the preferred system for gene silencing in the brain [50, 51].

Vector engineering has also been crucial so that AAVs acquire novel biomedically valuable properties through two main methods: rational design and

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directed evolution [52]. The first approach aims to improve viral vectors based on previous knowledge of capsid structure and delivery mechanisms. In particular, the

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discovery that phosphorylated tyrosine residues promote ubiquitination and consequent proteasomal degradation led to site-directed mutagenesis replacing

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tyrosines on the capsid surface by phenylalanine residues. The final result was a

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mutant AAV capsid with 30-fold higher transgene expression in vivo [53]. Another possibility is the combination of capsid domains from different serotypes to generate

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hybrid vectors with considerable characteristics from each original serotype [54]. For instance, the mosaic virus AAV2/1 has been generated using capsid components from both serotypes, in order to combine the AAV1 tropism and the simple purification

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method of AAV2 [55]. Directed evolution, on the contrary, starts with a viral library created by random mutagenesis on capsid genes or by combination of serotypes through DNA shuffling. Libraries are then subjected to multiple rounds of selection to obtain novel variants with desired properties. This technique was successfully used to isolate AAV capsids with significant resistance against neutralizing antibodies, for example [56, 57].

2.4. rAAVs in CNS clinical studies

Due to their unique properties, AAVs have been extensively investigated in the context of human gene therapy. The establishment of an AAV1-based strategy for lipoprotein lipase deficiency treatment was a major breakthrough in the field, leading 10

ACCEPTED MANUSCRIPT to the first commercially available gene therapy product in Europe in 2012, under the name of Glybera [58]. Moreover, several clinical trials already reported stable FIX (coagulation factor IX) expression in hemophilia B patients following AAV-based

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therapy [59]. These encouraging results opened the way to extensive investigation

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exploring the clinical applications of AAV gene delivery, namely in CNS disorders, which

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are the focus of the present review.

As depicted in Table 2, AAV administration has been tested in motor neuron disorders (Spinal Muscular Atrophy type 1), lysosomal storage disorders (Batten and Sanfilippo syndrome), a neurotransmitter disorder (Aromatic L-amino Acid

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Decarboxylase (AADC) Deficiency), a glycogen storage disorder (Pompe disease), neurodegenerative disorders (e.g. Parkinson disease), and several eye disorders (e.g.

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Age-Related Macular Degeneration, Leber Congenital Amaurosis). Concerning the latter group, the most commonly used strategy involves AAV2

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subretinal or intravitreal administration. Regarding the remaining disorders,

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intraparenchymal AAV2 injection is still the most commonly used method, although other serotypes (AAVrh.10, AAV1, AAV5 and AAV9) and delivery routes (intravenous

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and intramuscular) are now emerging as potential alternatives.

Table 2 – Summary of clinical trials for CNS disorders using rAAVs Disease classification

Disease

Serotype

Motor neuron diseases

Spinal Muscular Atrophy type 1 (SMA)

AAV9

AAV2 Lysosomal storage disorders

Batten AAVrh.10 MPSIIIB (Sanfilippo syndrome B)

Glycogen storage disorders

Route and local of administration Intravenous Intraparenchymal Direct administration into the cortex Intraparenchymal

Transgene

Clinical Trial (Phase)

Identifier

SMN

Phase I

NCT02122952

CLN2

Phase I

NCT00151216

CLN2

Phase I and II

NCT01414985 NCT01161576

Intraparenchymal

NAGLU

AAV9

Intramuscular Injection in the tibialis anterior muscle

AAV1

Intramuscular Intradiaphragmatic

Phase I and II

ISRCTN19853672 http://www.isrctn.co m

GAA

Phase I

NCT02240407

GAA

Phase I and II

NCT00976352

AAV5

Pompe

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GAD

AAV2

Intraparenchymal Bilateral stereotactic injections into the putaminal region

AAV2

Intraparenchymal Bilateral delivery by convection-enhanced delivery (CED) to the putamen

AAV2

Intraparenchymal Injection into the striatum

AAV2

Canavan

AAV2

AAV2

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Age-Related Macular Degeneration (AMD) Leber Congenital Amaurosis (LCA)

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AAV2

AAV2

X-linked Retinoschisis

AAV8

Choroideremia Eye disorders

Intraparenchymal Bilateral injection into putamen Intraparenchymal Bilateral infusions into the frontal, periventricular and occipital lobes

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Leukodystrophies

Aromatic L-amino Acid Decarboxylase (AADC) Deficiency

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Monoamine neurotransmitt er disorders

AAV2 Leber's Hereditary Optic Neuropathy (LHON)

AAV2

Achromatopsia

AAV2

NTN

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Parkinson

Subretinal

Subretinal

Subretinal

Intravitreal Intravitreal Intravitreal

Subretinal

T

AAV2

Intraparenchymal Bilateral surgical infusion into the subthalamic nucleus

Phase I and II

Phase I and II

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NGF

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Idiopathic neurodegenera tive disorders

AAV2

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Alzheimer

Intraparenchymal Stereotactic injections into the basal forebrain

GDNF

AADC

AADC

ASPA

NCT00087789 NCT00876863

NCT00195143 NCT00643890 NCT01301573

Phase I and II

NCT00252850 NCT00400634 NCT00985517

Phase I

NCT01621581

Phase I and II

NCT00229736 NCT02418598 NCT01973543

Phase I and II

NCT01395641

(McPhee, Janson et al.

Phase I

2006)

Phase I and II

NCT01494805 NCT01024998

Phase I, II, III

NCT00749957 NCT00999609 NCT01208389 NCT00516477 NCT00643747

REP1

Phase I and II

NCT01461213 NCT02553135 NCT02407678 NCT02077361 NCT02341807 NCT02671539

RS1

Phase I and II

NCT02317887

RS1

Phase I and II

NCT02416622

ND4

Phase I and III

NCT02161380 NCT02652780 NCT02652767

CNGB3

Phase I and II

NCT02599922

sFlt-1

RPE65

Data obtained from clinicaltrials.gov (on 05-31-2016) Abbreviations: NG - Nerve Growth Factor; GAD - Glutamate decarboxylase; NTN Neurturin; GDNF - Glial Derived Neurotropic Factor; ASPA- Aspartoacylase; AADC Aromatic L-Amino Acid Decarboxylase; CLN2 - Tripeptidyl Peptidase-I; NAGLU - α-Nacetylglucosaminidase; SMN - Survival of Motor Neuron; GAA - Acid alpha-glucosidase; 12

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sFlt-1 - Soluble fms-like tyrosine kinase-1; REP1 - Retinal pigment epithelium-specific protein; RS1 - Retinoschisin 1; ND4 - NADH dehydrogenase 4; CNGB3 - cyclic nucleotide gated channel beta 3

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3. Routes of rAAV administration to the CNS

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Intraparenchymal injection has been the most commonly used route of administration of AAV vectors to the CNS by our group and others [60, 61], as well as in the majority of clinical studies (Table 2). The direct delivery into the brain parenchyma

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circumvents the blood-brain barrier (BBB), but leads to poor vector spread (1-3 mm), and as a result transgene expression is limited to the site of injection [62]. This is an

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important limitation for many neurodegenerative disorders that affect large regions of the CNS, such as lysosomal storage disorders or Alzheimer’s disease. Additionally, each

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injection requires a craniotomy and general anesthesia, associated with the risk of hemorrhaging and pathogen contamination. Accordingly, a clinical study for

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surgical procedure.

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Parkinson’s disease (NCT00400634) has reported serious side effects related to this

Taking this into account, the ideal alternative would correspond to a noninvasive procedure that enables widespread vector distribution in the CNS. A possible solution is to infuse AAV vectors into the cerebrospinal fluid (CSF). This can be done

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through intracerebroventricular (ICV), intra-cisterna magna (ICM) or lumbar intrathecal injections (IT), as illustrated in Figure 3, with the latter being the least invasive and consequently the most attractive for clinical applications. Moreover, intramuscular injection is considered a suitable approach to motor neuron diseases treatment. In fact, some AAV vectors can be taken up by nerve terminals and undergo retrograde transport to motor neurons soma in the spinal cord [63]. Finally, intravascular injection is also a promising alternative, being generally divided into intra-arterial (e.g. intracarotid or intrafemoral) and intravenous (IV) routes, as depicted in Figure 3. Although intravascular administration is the least invasive route, it is entirely dependent on whether a particular AAV vector is able to cross the blood-brain barrier (BBB) [64].

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ACCEPTED MANUSCRIPT The BBB incorporates endothelial cells that are connected by tight junctions and surrounded by pericytes and unsheathed in astrocytic end feet [65]. This barrier restricts the movement of macromolecules, including viral vectors, from the blood into

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the CNS. Therefore, recent research has developed new methods to circumvent this

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barrier and increase transgene expression in the brain after intravascular rAAV

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delivery. Transient BBB permeabilization through osmotic disruption, for example, might be useful for that purpose. Systemic infusion of hyperosmotic mannitol is a potential approach since it has been used clinically to reduce brain swelling after traumatic injury. Fu et al. [66] have studied the impact of this method on AAV2

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distribution throughout the mouse brain upon systemic injection, having observed an increase in neuronal and glial transduction due to mannitol infusion. Similarly, Burger

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et al. [67] demonstrated its impact on the total number of transduced cells and on vector striatal distribution, showing a 400% and 200% increase, respectively.

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Moreover, according to a subsequent study by McCarty et al. [68] the effect of

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mannitol in rAAV CNS entry potentiates its therapeutic action in neurological diseases after IV injection. However, it is important to state that this method generates an

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increased influx of several molecules to the brain and presents several side-effects (e.g. hypotension, pulmonary congestion, electrolyte imbalance) [69, 70]. Alternatively, focused ultrasound in combination with microbubbles (FUS) is a

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non-invasive strategy that locally and transiently disrupts the BBB in rodents and nonhuman primates [71, 72]. Additionally, FUS can be complemented with magnetic resonance imaging (MRI) in order to observe and guide BBB opening [73]. This technique presents important advantages when compared to mannitol infusion, as it provides a better control of BBB permeability and the possibility of targeting specific brain regions. According to recent studies, MRI-guided FUS has already shown encouraging results following IV injection of AAV2, AAV2/1 and AAV9 in rodents, by inducing stable transgene expression in the brain, even when using low AAV titters [74]. Moreover, Marquet et al. [75] proved that FUS-mediated BBB disruption can be translated to non-human primates and Downs et al. [76] demonstrated the safety profile of this technique.

14

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ACCEPTED MANUSCRIPT

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Figure 3 – Possible routes of rAAV administration to the CNS: 1) Intraparenchymal injection; 2) Administration into the CSF: intrathecal, intra-cisterna magna and intracerebroventricular injections; 3) Intramuscular administration; 4) Intravascular delivery: intravenous, intracarotid and intrafemoral administration.

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4. Non-invasive gene delivery to the CNS using AAV9

Despite all the advantages of systemic AAV delivery, this method has been hindered by the fact that most serotypes cannot circumvent the BBB. As indicated in Table 1, IV administration does not induce an efficient CNS transduction for most of the known serotypes. Consequently, this delivery route often requires pharmacological (e.g. mannitol) or physical strategies to disrupt the barrier, which may induce adverse effects and hamper future clinical progresses, as described above. Therefore, the discovery that a particular serotype, AAV9, has the natural ability to bypass the BBB has expanded the applications of intravascular AAV administration in CNS gene therapy. In fact, Foust [77] and Duque et al. [78] demonstrated that AAV9 circumvents this barrier in both neonatal and adult mice, 15

ACCEPTED MANUSCRIPT leading to widespread CNS gene expression. The mechanism AAV9 uses to cross the BBB is still unknown. According to Gray et al. [79], mannitol co-administration, which is supposed to disrupt BBB, does not significantly enhance AAV9 CNS transduction in

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mice. These results indicate that the vector does not passively slip through the tight

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junctions between endothelial cells, but is actively transported across BBB. According

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to Manfredsson [80], AAV9 might interact with other receptors besides terminal Nlinked galactose, which normally mediate the transport of several molecules from the blood to the brain (e.g. monocarboxylate transporter 1 for lactate (MCT1) or glucose transporter GLUT1). Although the capsid aminoacids that dictate this AAV9 property

regions (VRs) as probable candidates.

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are still unknown, DiMattia et al. [81] pointed out residues in nine variable surface

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According to recent reports, this BBB-crossing property is not restricted to AAV9. Several other serotypes have also shown CNS transduction after systemic

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injection, both in neonatal and adult rodents, with varied transduction efficiencies.

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Based on these results, rAAVrh.10, rAAVrh-8, rAAVrh.39, rAAVrh.43, rAAV7 and rAAV8 are also considered potential candidate vectors for CNS non-invasive gene delivery [35,

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36]. According to Zhang and co-workers, AAVrh.10 holds great promise, due to its extensive distribution and stable transgene in the brain after IV injection in neonatal mice. On the other hand, when systemically injected in adult mice, rAAVrh.8 stands

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out due to its tropism. Besides targeting CNS clinical relevant regions, this serotype displays reduced peripheral tissue dissemination [36]. However, AAV9 has been the most widely studied vector for IV delivery, since this serotype is among the best performers in both studies, showing high transgene expression and widespread transduction throughout the CNS in neonatal and adult mice.

4.1. Neonatal versus adult IV injection

Although able to cross the BBB independently of the animal age, AAV9 exhibits different transduction profiles in the CNS after IV administration to newborn or adult subjects. Table 3 summarizes the principal studies exploring the outcomes of AAV9-IV injection in different animal and time points.

16

ACCEPTED MANUSCRIPT Upon post-natal IV injection in mice, Foust et al. [77] observed transduction in neurons and astrocytes in multiple brain regions and in motor neurons (MNs) in the spinal cord. Subsequent investigation has consistently confirmed that AAV9-IV

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administration to newborn mice induces efficient neuronal transduction. Wang et al.

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[82] have documented a maximal transduction of 78% spinal lower MNs. The authors

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also detected significant transgene expression levels in the brain, both in neurons and glia, which according to Miyake et al. [37] are stable for at least 18 months. In conclusion, all these studies reported the same pattern in newborn mice: the transduction of neurons and astrocytes in the brain and a significant preference for

NU

MNs in the spinal cord [83]. On the contrary, Foust et al. [77] concluded that in adult mice there is a predominant astroglial transduction in the brain and spinal cord, only

MA

with occasional transduced neurons. However, subsequent studies have achieved significant neuronal transgene expression following AAV9-IV injection during

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adulthood, contradicting the previous results. Concerning the spinal cord, Duque et al.

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[78] reported a successful transduction of MNs in adult subjects using this delivery system. In fact, the authors reported a long-term transgene expression of at least 5

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months in 28% of MNs. This finding was successfully translated to cats, where spinal cord MNs were efficiently transduced not only in neonate but also in adult subjects. The same outcome was reported by Gray and co-workers following AAV9 IV injection

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in adult mice [79]. Besides observing efficient MN transduction in the spinal cord, the authors also detected significant expression levels in the brain, both in neurons and glia, although in a low percentage of cells. Surprisingly, for specific viral titters (1.25 × 1012, 1 × 1013, and 8 × 1013 vg/kg), the vector actually transduced twice as many neurons as astrocytes throughout the CNS, which is incompatible with previous results from Foust et al. [77]. In conclusion, the literature shows no clear consensus in what concerns neuronal transduction efficiency in adult mice. Nevertheless, all studies point towards the same conclusion: neuronal transduction efficiency declines with age. Accordingly, Duque et al. documented a reduction from 39% to 15% MNs in cats, when comparing injection in newborn and adult subjects. Likewise, Foust et al. [84] defined the period between P1 and P10 as the ideal time window to target spinal MNs. Finally, Miyake [37] et al. observed a similar efficiency decline in the brain. Taking all of this into account, earlier stages of development are considered the most suitable periods 17

ACCEPTED MANUSCRIPT to induce therapeutic effects directed to mouse neurons. On the other hand, this reduction is coupled with an increase in glial transduction, which might be useful to correct CNS disorders affecting this cell population [85] [86].

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Several hypotheses have been proposed to explain why neuronal/astrocytic

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preferential transduction is age dependent. According to Lowenstein [87], the type of

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cells surrounding blood vessels and the brain extracellular space are subjected to some alterations during development, leading to different transduction patterns. Thus, in neonates, AAV9 encounters a lax extracellular matrix after crossing the endothelial barrier, explaining why it is able to diffuse and transduce neurons. Additionally,

NU

neurons are more predominant than glia in neonatal brain, favoring this transduction profile. In contrast, in adult mice, endothelial cells are surrounded by astrocytic

MA

endfeet, possibly explaining why AAV9 preferentially transduces astrocytes and endothelial cells.

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Subsequently, several authors assessed whether the same outcome occurs in

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non-human primates (NHPs). In this context, it has been shown that AAV9 bypasses the BBB and efficiently transduce neurons after IV delivery to neonatal rhesus

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macaques [84]. Moreover, reports from Dehay [88] and Gray [79] consistently match the results obtained in mice: widespread neuronal transduction in the brain following injections at P1, in contrast to glial preference when administration is performed in

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juvenile NHPs. However, this progressive decline in neuronal versus glial transduction has been contradicted by Bevan and co-workers [89]. These authors tested the same approach in cynomolgus macaques, from birth to 36 months of age, leading to the conclusion that AAV9 transduction pattern is maintained over time and consists on preferentially glial cells throughout the brain and MNs in the spinal cord. According to the authors, the discrepancy between rodents and NHPs might derive from differences in the timing of gliogenesis. The first observation might eventually hamper the therapeutic efficacy of AAV9 in disorders affecting neurons in the brain. At the same time, it emphasizes the utility of AAV9 systemic administration for gene therapy targeting astrocytes and microglia. Finally, the successful transduction of MNs in NHPs regardless of the injection time point highlights the possible therapeutic impact of this method in human motor neuron diseases. In fact, the results in NHPs suggest the therapeutic time window may be longer in humans than rodents. 18

ACCEPTED MANUSCRIPT In summary, the previously described studies were a major breakthrough in the field since: i) they consistently corroborated the possibility of CNS gene transfer through a non-invasive systemic delivery route and ii) they revealed AAV9 natural

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tropism to CNS upon IV injection using different models and administration times.

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Nevertheless, these studies exhibited low reproducibility and controversial results. This

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might also be explained by differences in promoter selection, viral doses (as indicated in Table 3) and viral vector production/purification methods. Therefore, further investigation with well-controlled and highly homogeneous vector formulations will be

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crucial to clarify the AAV9 CNS transduction profile after systemic administration.

19

ACCEPTED MANUSCRIPT Table 3 – Summary of the most relevant studies exploring AAV9-IV injection in different animal models at different time points. Animal model

Time of injection

Genome

Promoter

Viral Titter

Time postinjection

Cellular transduction in the brain

11

Mouse

P1

SC

CBA

4×10 vg/animal

[82]

Rat

P1

SS

CAG

2x10 vg/animal

[83]

Mouse

P1

SS and SC

CMV

4x10 vg/animal

1 month

[37]

Mouse

SS

CAG

1.5×1011 – 10 12 vg/animal

18 months

P1, 5, 14

Mouse

P70

SC

[78]

Mouse

6 weeks

SC

Mouse

8 -12 weeks

[78]

Cat

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3. Cats

SC

P2

SC

[84]

[89]

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4. Non-human primates

Cynomolgus macaque

P1

Cynomolgus macaque

P1-P90

SC

SC

CBA

CBA

Predominantly astrocytes in different brain regions + Purkinje cells Mainly neurons, but also astrocytes (decrease over time)

Scarce MN transduction; Mainly astrocytes

3 × 1011 - 2 × 1012 vg/animal

2-4 weeks

_

Transduction of MNs and astrocytes

2.5 × 1010 1.6 × 1012 vg/animal

4 weeks

Neurons and astrocytes (neuronal predominance in some regions)

Transduction of MNs and astrocytes

1.5 × 1012 vg/animal

1 × 1014 vg/kg 1-3 × 1014 vg/kg

_

15 days a y s

1.2 × 1012 vg/animal

7 weeks

Neurons and astrocytes in several brain regions

2-7 weeks

CMV

CMV

High transduction of MNs

Only localized neuronal expression in few regions; Mainly astrocytes

CBA

CBA

4-12 weeks

11

4×10 1012 vg/animal

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[77]

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2. Adult rodents

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11

Neurons and astrocytes in several brain regions

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[77]

12

10 or 21days

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1. Neonatal/Infant rodents

[79]

Cellular transduction in the spinal cord

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Study

21 days

21-24 days

34-39% MN

15% MN

_

Transduction of MNs

Glial cells

Transduction of MNs

2.7 ×1013 vg/kg

3 years [79]

Rhesus macaque

3–4 years

SC

CBA

1 × 1013 vg/kg

4 weeks

Mostly glia

Mostly glia

[88]

Rhesus macaque

P1

SC

CMVie

1012-1015 vg/kg

2 months

Widespread neuronal targeting

_

[90]

Cynomolgus macaque

?

SC

CBA

3.0x1013 vg/kg

21 days

Predominantly astrocytes, but also neurons

_

Abbreviations: SS – single-stranded; SC - self-complementary; vg - viral genome; CMV – cytomegalovirus promoter; CMVie - cytomegalovirus immediate early synapsin intron promoter; CBA- chicken β-actin promoter; CAG - hybrid promoter comprising 20

ACCEPTED MANUSCRIPT the CMV immediate-early enhance, CBA promoter and CBA intron 1/exon 1; vg – viral genomes; MN – motor neuron

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4.2. Intrauterine administration

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In the case of neurodegenerative monogenic diseases associated with perinatal

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mortality (e.g. Gaucher Disease), gene delivery in utero may be necessary to completely prevent irreversible neuronal damage. However, few studies have explored intrauterine delivery route, possibly due to the technical difficulties of this

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procedure.

Rahim et al. [83] recently performed a direct comparison between IV of AAV9 to fetal and neonatal mice. As a result, the authors observed a robust and widespread

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gene delivery to the CNS after in utero delivery, with a predominant neuronal tropism. On the contrary, post-natal AAV injections induced a preferential astrocytic

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transduction. Mattar et al. also demonstrated efficient neuronal transduction after in

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utero delivery of AAV9 in non-human primates [91]. Nevertheless, this method presents significant practical concerns related to prenatal diagnostic techniques and

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the risks associated with the treatment [92]. Taking all of this into account, newborn IV administration is still considered a preferential option.

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4.3. Preclinical studies for CNS using AAV9

The discovery of AAV9 ability to cross BBB was the starting point for an extensive investigation regarding its therapeutic effect in a wide range of CNS disorders. Table 4 summarizes the main studies in the field, in which intravenous is the predominant delivery route. In general, AAV9 is used to restore a faulty gene, as in the case of lysosomal storage disorders and Spinal Muscular Atrophy type 1; to silence a dominantly inherited mutant allele, as in Huntington disease; or to introduce a disease-modifying gene that might alleviate neuropathology, as investigated for Alzheimer’s disease. Theoretically, the most suitable candidates for vascular AAV9-mediated gene therapy, considering its natural properties, would be disorders: i) with a defined 21

ACCEPTED MANUSCRIPT genetic cause, so that it is possible to directly target the affected gene; ii) presenting an early-onset, favoring vector administration during childhood, when neuronal tropism is likely more robust; iii) affecting both CNS and peripheral organs, in order to

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fit the natural AAV9 biodistribution. Motor neuron diseases

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Since AAV9 transduces spinal MNs very efficiently, numerous preclinical studies have focused on motor neuron disorders, particularly Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral Sclerosis (ALS). SMA is characterized by a loss or

NU

mutation on the telomeric copy of the ‘survival of motor neuron’ gene, termed SMN1. Although the centromeric form (SMN2) is maintained, it does not generate sufficient

MA

quantities of full-length SMN protein, which is crucial for the assembly of ribonucleoprotein complexes. In fact, a C-to-T substitution interferes with splicing, leading to exon 7 skipping and consequently to the production of a truncated and

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unstable form of SMN (SMNΔ7). The reduction in SMN protein levels ultimately leads

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to MNs degeneration, progressive muscle weakness, atrophy and paralysis. SMA is

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currently considered the main genetic cause of infant mortality and no effective treatment is still available. Therefore, one of the most attractive approaches for SMA treatment is to reintroduce SMN1 using viral vectors. In this context, several studies have investigated the impact of AAV9 encoding SMN, particularly through systemic

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administration in neonatal transgenic mice. Firstly reported by Foust et al. [84], this therapy resulted in significant improvements regarding motor function, neuromuscular physiology and life span, by increasing SMN levels in the spinal cord, brain and muscles. However, the therapy has maximal benefit in a particular developmental period, showing little effects when the injection is performed at post-natal day 10. Contemporary studies confirmed the benefic effects of AAV9-SMN1 delivery in SMA neonatal mice [93, 94], leading to a maximal mean life expectancy of 160 +/- 39 days, a significant improvement when comparing to the untreated animals life span of 14 days. Alternative delivery paradigms have also been explored, such as intra-CSF and intramuscular injections. Intramuscular AAV9-SMN delivery has been suggested as a simple and non-invasive delivery route, resulting in axonal retrograde vector transport to the MNs that are connected to the injected muscles. As recently reported [63], this 22

ACCEPTED MANUSCRIPT type of administration is able to induce an extensive gene transfer in MNs and peripheral organs, both in neonatal and adult mice. Therefore, this treatment is able to ameliorate SMA mice symptoms, leading to an increase of life span from 12 to 163

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days. Additionally, Glascock et al. [95] have tested the impact of

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intracerebroventricular injections in a severe SMA mouse model, having observed

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improvements in body weight and life span. When compared to intravenous injection using the same AAV9 dose, ICV administration has proved to be more efficient. As confirmed by Meyer et al. [96], ICV administration is able to produce the same outcomes as IV administration, but using a 10 times lower AAV dose. Despite the clear

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benefits of intra-CSF delivery, this type of administration may induce suboptimal expression levels in the peripheral tissues. Since this delivery route is restricted to the

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CNS, AAV9 will efficiently target spinal cord MNs, which are the primary SMA pathological target. However, according to some reports, SMA mouse models present

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defects in other tissues, including the muscle, heart and pancreas [97]. Taking this into

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account, intravenous injection could be the ideal alternative to achieve central and peripheral SMN restoration and, consequently, complete correction of this

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multisystem disorder.

Notably, several experiments exploring AAV9 ability to transduce spinal cord MNs and other tissues in large animal models have already been performed, after

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systemic and intra-CSF administration, providing confidence for translation to human patients [84] [89]. Very recently, AAV9-SMN1 intrathecal delivery has also successfully corrected the phenotype of a pig SMA model [98]. All of these successful reports culminated in the first clinical trial approved for the test of AAV9 in neurodegenerative disorders (NCT02122952) (see Table 2). This Phase I dose escalation study (1013-1014 vg/kg) is currently recruiting type 1 SMA patients, namely infants with less than 9 months. The main goal is to test the efficiency and safety of a single scAAV9.CB.SMN intravenous delivery. Therefore, alterations in hematology, serum chemistry, urinalysis and immunologic responses will be assessed during a 2-year period. This revolutionary step might be crucial to evaluate the therapeutic impact directly on humans, to identify possible adverse effects and define ideal viral doses for future treatments.

Neurodevelopmental disorders 23

ACCEPTED MANUSCRIPT Neurodevelopmental disorders, such as Rett Syndrome, meet the abovementioned requirements for vascular AAV9-mediated gene therapy. This syndrome’s

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genetic cause is known to involve loss-of-function mutations in methyl-CpG binding

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protein 2 (MeCP2). Thus, Garg et al. [99] demonstrated that AAV9 is able to provide physiological levels of MeCP2 throughout the brain, reverting the symptoms in female

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RTT mice after systemic injection. Additionally, the fact that AAV9 tropism is not neuronal-specific might be an advantage in this case, since MeCP2 glial expression has

Lysosomal storage disorders

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proved to be sufficient to ameliorate RTT symptoms [100].

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AAV9 has also shown impressive therapeutic results for lysosomal storage disorders (LSD). These diseases are triggered by an accumulation of different

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metabolites in lysosomes due to enzymatic defects; they are multi-systemic and roughly 60% of cases involve the CNS. Due to the BBB, enzyme replacement and

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previous gene therapy methods have not resulted in whole-body correction in many

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cases.

According to Table 4, extensive research has been focused on mucopolysaccharidoses (MPS), a group of LSDs characterized by defects in the degradation of glycosaminoglycans (GAGs). For instance, in MPSIIIB or Sanfilippo

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syndrome, a single IV injection restored enzymatic activity in adult mice, providing stable correction of lysosomal storing in the CNS and other tissues, behavior improvements and survival extension [101]. Subsequently, Murrey et al. [42] tested the same approach in wild-type cynomolgus monkeys, successfully increasing enzymatic activity throughout the body. Importantly, safety aspects were also explored, with no reports of adverse events or cytotoxic T lymphocyte (CTL) responses, opening the way for future clinical trials. Similar improvements were achieved for other mucopolysaccharidoses (MPSI, MPSIIIA, and MPSVII). Moreover, several studies have focused on a different class of LSDs, called Sphingolipidoses, which are characterized by defects on sphingolipid metabolism (e.g. GM1-gangliosidosis, Sandhoff Disease, Multiple Sulfatase Deficiency and Metachromatic leukodystrophy), also showing successful results as described in Table 4. 24

ACCEPTED MANUSCRIPT

Glycogen storage diseases Current research has focused on Pompe disease, a genetic disorder induced by

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defects in the lysosomal degradation of glycogen due to insufficient levels of acid

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alpha-glucosidase (GAA). Pompe disease was thought to be the result of the metabolic

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defect in muscles, but recent studies have shown that muscle dysfunction is also caused by motor neuron disease. Enzyme replacement therapy (ERT) is an accepted treatment for Pompe disease. However, this strategy is not able to completely correct the disorder, only attenuating the skeletal muscle weakness, probably because it

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cannot target the CNS. Therefore, the ideal alternative would be to simultaneously reach the myocardium, skeletal muscle and neurons. For that purpose, Flark et al.

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tested the impact of AAV9-GAA intrapleural injection, leading to increased cardiac and respiratory function in adult Gaa−/− mice [102]. In addition, one of the major

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hallmarks of this disorder concerns neuromuscular junction (NMJ) defects, which

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according to Todd et al. [103] can be corrected by a single AAV9-GAA intramuscular injection. In fact, this treatment promoted muscular glycogen clearance and

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consequently normalized NMJ structure and function. All together, these findings culminated in a novel clinical trial (NCT02240407) that aims to evaluate the safety profile and therapeutic impact of intramuscular AAV9 administration in late-onset

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Pompe disease.

Polyglutamine diseases AAV9 systemic delivery has been also investigated in the context of polyglutamine disorders, caused by CAG triplet expansions on the causative genes. Regarding Huntington disease, Dufour et al. [104] have tested a gene silencing approach targeting specifically mutant huntingtin (mHTT). In this case, AAV9 encoding the RNAi construct was administrated in the intrajugular vein of 3-week-old mice, causing a reduction in mHTT levels in multiple brain regions and peripheral tissues. Although able to prevent neuropathological changes, this treatment had no significant impact on motor deficits, stressing the importance of optimization techniques in future investigation. Besides gene silencing, a possible therapeutic strategy to polyglutamine disorders consists on preventing protein aggregation, as tested in a transgenic mouse 25

ACCEPTED MANUSCRIPT model of Machado-Joseph Disease by Konno et al. [105]. In this study, the authors used AAV9 vectors encoding CRAG (collapsin response mediator protein-associated molecule-associated guanosine triphosphatase), a GTPase that contributes to the

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degradation of stress proteins. Consequently, this treatment was able to reduce

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ataxin-3 aggregation, a major hallmark of the disease, and improve dendritic

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differentiation. However, the authors have not performed behavioral tests, leading to no conclusions regarding the therapeutic effect of AAV9-CRAG administration. Idiopathic neurodegenerative diseases

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Gene therapy for idiopathic disorders, such as Parkinson and Alzheimer’s disease (AD) is more challenging, since the primary cause of non-familial cases that

MA

represent >90% of cases is still unknown. As indicated in Table 4, the most common strategies consist on inducing neuroprotection or ameliorating well-established

D

disorder hallmarks. For example, Iwata et al. [106] performed an intracardiac

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administration of AAV9 encoding neprilysin (an enzyme involved in brain amyloid-beta catabolism) in a mouse model for AD. Therefore, the global transgene delivery in the

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brain led to a decrease in Aβ oligomer levels, consequently alleviating the characteristic learning and memory impairments of this disease.

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Overall, all these findings demonstrate the versatility of AAV9 injection in CNS gene therapy, as it induces positive effects using different therapeutic strategies, administration routes or injection time points. As depicted in Table 4, intravenous administration has been the most commonly used delivery system due to its noninvasive nature and resulting widespread gene transfer to the CNS.

26

ACCEPTED MANUSCRIPT Table 4 – Description of preclinical studies using AAV9 for CNS gene therapy.

Spinal muscular atrophy (SMA)

Loss of SMN1 (Motor neuron death)

Time of injection

Delivery route

Mouse Neonatal

P1

IV

Mouse Neonatal

P0/ P1

Mouse Neonatal Pig

Mouse Neonatal

D

Mutations in IGHMBP2 (Motor neuron death)

TE

Motor neuron diseases

Spinal muscular atrophy with respiratory distress type 1 (SMARD1)

CE P

Mouse Neonatal Juvenile

Familial: Mutations in SOD1 (Motor neuron death)

AC

Amyotrophic lateral sclerosis (ALS)

Sporadic: downregulation of ADAR2 (Motor neuron death)

Neurodevelopmental disorders

Rett Syndrome

Mutations in MECP2 (Neurodevelopmental problems)

Mouse Adult

Rat Adult

Intra-CSF (ICV)

Intra-CSF (ICM)

P1

IV

P1

Earlyonset model: P1, P21, P85

IV

Mouse Adult

9–15 Weeks

Mouse Neonatal

P1

Ref

Survival of motor neurons, rescue of neuromuscular physiology and life span

[84] [93] [94] [95]

Improvements in muscle physiology and survival extension More significant effects than IV injection

[95, 107]

[63]

Improvements in the phenotype, electrophysiology and pathology

[98]

siPTEN

Phenotype improvement and survival extension

[108]

IGHMBP2

Motor improvements, neuromuscular physiology normalization and life span extension

[109]

Delay of disease onset, behavioral improvements and survival extension for P1-injected animals. More modest effects for P21 and P81. SOD1 shRNA

Intraparenchymal

IV

Result

Extension in life span, phenotypic and weight loss alleviation

IV

Late onset model: P215

P70

SMN

IM

P5 P33-36

MA

Mouse Neonatal

P1

Transgene

T

Animal model Age

IP

Genetic cause (Neuropathology)

SC R

Disease

NU

Disease classification

ADAR2 (SynI promoter)

Intraparenchymal

[110]

Slow disease progression, survival extension Spinal motor neurons survival, maintenance of NMJs , delay of disease onset and lifespan expansion

[111]

Prevention of motor dysfunction and neuronal death. Behavioral improvements

[112]

Improvement in survival and phenotypic alleviation [113] MeCP2

Mouse Juvenile

4-5weeks

IV

Modest effect in survival, partial reversion of synaptic defects

27

ACCEPTED MANUSCRIPT

MPS I

Cat

4–7 months

IV

Rescue of behavioral and cellular deficits

[99]

Intra-CSF (ICM)

Correction of CNS disease manifestations. Antibody responses against IDUA. No evidence of toxicity

[114]

Mutations in SGSH (Neurodegeneration and peripheral organ changes)

Mouse Adult

Mouse Adult

2 months

Mouse Adult

2 months

TE CE P

Mutations in GUSB (Neurological and peripheral problems)

AC

MPS VII (Sly syndrome)

GM1gangliosidosis (GM1)

GLB1 mutations (Neurodegeneration in the brain and spinal cord)

Sandhoff disease

Deficiency in HEXB (Neurodegeneration in the brain and spinal cord)

Multiple sulfatase deficiency (MSD)

Mutations in SUMF1 (Neurological and peripheral defects)

4–6 weeks

MA

D

Defects in NAGLU (Neurological and peripheral problems)

Lysosomal storage disorders

SGSH

Intra-CSF (ICM)

Mouse Juvenile MPSIIIB (Sanfilippo syndrome B)

2 months

NU

MPSIIIA (Sanfilippo syndrome A)

SC R

IV

IDUA

T

Deficiency of IDUA (Neurological and peripheral problems)

10-12 months

IP

Mouse Adult

P3

IV

NAGLU

Intra-CSF (ICM)

Intraparenchymal

IV

GUSB

Dog P24

Mouse Juvenile

Mouse Neonatal Mouse Adult

Mouse Neonatal

6 weeks

Intra-CSF (ICM)

IV

Intra-CSF (ICV) or/ and IV

Correction of lysosomal storage defects, amelioration of astrocytosis and neurodegeneration. Improvement in behavioural performance and longevity.

[115]

[116]

[101]

Normalization of lysosomal physiology, resolved neuroinflammation, reversal of behavioral deficits and extended lifespan.

[117]

Lysosomal storage correction in the brain

[118]

Reduction in GAGs accumulation and inflammation. More significant effects for intra-CSF injection

[119]

Phenotypic amelioration and extension in lifespan

HEXB

Reduction in GM2 ganglioside storage, improvements in motor activity and longevity. More impact on neonatal than adults.

SUMF1

The combined treatment is more efficient, leading to: global activation of sulfatases, decrease of inflammation and

6 weeks

P1-2

Correction of wholebody GAG accumulation, behavioral improvements and survival extension. Effects dependent on the dose

βgal

P1-P2 IV

Correction of CNS and peripheral GAG accumulation, reduction in neuroinflammation and lifespan extension

28

[120]

[121]

[122]

ACCEPTED MANUSCRIPT behavioral improvements

Pompe Disease

Mutations in GAA (Disruption of neuronal and muscle homeostasis)

Mouse Adult

3 months

Mouse Adult

2 months

Mouse Juvenile Adult

Polyglutamine diseases

Huntington Disease

MachadoJoseph Disease

Parkinson Disease

Alzheimer’s Disease

1, 9, 15 months

MA

6-8 weeks

Mouse Juvenile

IP

Intrapleural

IM

Intra-CSF (ICV)

P1-2

IV

Intra parenchymal

Mouse Neonatal

Degeneration of dopaminergic neurons

Rat Adult

?

Mouse Adult

7–9 months

Accumulation of amyloid-β in the brain

IV

Glycogen clearance, improvements in cardiac and respiratory functions

[123]

[102]

[124]

Improvements in NMJ structure and function. Early treatment is more effective

[125]

ABCD1

Decrease in CNS VLCFA accumulation only for IV treatment

[126]

mHTT RNAi

Prevention of central and peripheral pathology and weight loss. No behavioral improvements.

[104]

CRAG ( MSCV promoter)

Prevention of mutant ataxin-3 aggregation, improved dendritic differentiation.

[105]

EPO

Neuronal protection against 6-OHDA-induced toxicity, behavioral improvements

[127]

NEP (SynI promoter)

Decrease in Aβ accumulation and alleviation of cognitive dysfunction

[106]

GAA

IV

IV

Correction of biochemical and neurological Abnormalities. Behavioral improvements

Glycogen clearance in motor neurons. Improvement in body weight

IM

3 weeks

Mutation in MJD1 (Inclusion formation and neurodegeneration)

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Idiopathic neurodegenerative diseases

Mutation in HTT (Inclusion formation and neurodegeneration)

ASA

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X-ALD

Mouse Juvenile

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Mutations in ABCD1 (Elevation of VLCFA levels, neurodegeneration)

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Peroxisomal disoders

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P0-P1

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Glycogen storage diseases

Mouse Neonatal

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Metachromatic leukodystrophy (MLD)

Mutations in ASA (Myelin degradation in the Nervous System)

Abbreviations: MPS – Mucopolysaccharidosis; X-ALD - X-linked Adrenoleukodystrophy; MECP2- methyl-CpG binding protein 2; SMN1 - Survival of Motor Neuron; SOD1 superoxide dismutase 1; IGHMBP2 - Immunoglobulin Mu Binding Protein 2; ADAR2 RNA-editing enzyme adenosine deaminase acting on RNA2; IDUA - α-l-iduronidase; SGSH - N-sulphoglucosamine sulphohydrolase (sulfamidase); NAGLU - α-Nacetylglucosaminidase; GUSB - Beta-glucuronidase; GLB1 - galactosidase beta 1; HEXB beta-hexosaminidase; Sumf1 - sulfatase modifying factor 1 gene; ASA- arylsulfatase; GAA - acid alpha-glucosidase; ABCD1- ATP-binding cassette transporter; VLCFA - Very Long Chain Fatty Acids; HTT – huntingtin; IV – intravenous; IM – intramuscular; ICV – intracerebroventricular; ICM – intra-cisterna magna; si-PTEN - RNA interference against PTEN; PTEN - phosphatase and tensin homolog; SynI - synapsin I; β-gal - β29

ACCEPTED MANUSCRIPT galactosidase; MSCV - Murine stem cell virus; EPO - erythropoietin ; NEP – Neprilysin; GAG – glycosaminoglycan.

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4.4. Intravascular versus intrathecal administration

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Although AAV9-IV administration is considered a versatile technique, with important applications in a wide range of CNS disorders, recent investigation has identified potential disadvantages for this delivery modality. These include exposure to circulating antibodies and off-target transduction, possibly inducing a significant viral

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vector loss, as well as serious adverse effects. As predicted from AAV immunization studies in rodents [128, 129], the presence of anti-AAV neutralizing antibodies (NAbs)

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in monkeys greatly reduces transgene expression [79, 90]. Similarly, this might also occur in humans, where NAbs to AAV9 are found in 47% of individuals and thus likely

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to restrict the therapeutic use of AAV9 vectors to a subset of patients. Moreover, upon

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IV delivery the majority of AAV9 vector genomes are found in peripheral tissues including liver, heart and skeletal muscle [37], potentially resulting in toxicity. In

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addition, high vector doses (1015 viral genomes for a 70- to 80-kg human) are required to achieve therapeutic effects as only a small quantity of injected vector reaches the CNS [90]. Lastly, AAV9 transduction occurs preferentially in astrocytes in adults, as

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previously mentioned, possibly reducing the therapeutic effect in neurons [79]. Considering these possible drawbacks, AAV9 delivery to CSF has been proposed as a promising alternative. In fact, Snyder et al. [130] demonstrated that AAV9 intrathecal administration in mice induces a widespread transgene expression in spinal MNs, a major advantage comparing to intraparenchymal injections. On the other hand, this method is expected to reduce peripheral transduction and capsid neutralization by circulating antibodies, the major limitations of IV delivery. Therefore, Bevan [89] and Federici [131] explored the efficiency of AAV9 intrathecal injection in pigs. As a result, the authors reported a robust transduction of MNs in the spinal cord and restricted gene expression to the CNS. Moreover, Samaranch et al. [90] directly compared intravascular and intra-CSF AAV9 delivery routes in non-human primates. According to this study, although both administration routes generated similar distribution patterns, intra-CSF injection induced a greater CNS transduction. Furthermore, current research 30

ACCEPTED MANUSCRIPT validated these findings in cats after intracisternal delivery [132]. This study showed that AAV9 is able to transduce the majority of MNs in the spinal cord and presents low off-target distribution both in newborn and young cats.

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However, some of these conclusions are still controversial. For instance,

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Shuster et al. [133] observed comparable levels of peripheral transduction using IV and

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intrathecal injections, suggesting that a fraction of IT delivered vector is moved from the subarachnoid space to systemic circulation. Moreover, according to Samaranch et al. [90], the presence of moderate serum titers of anti-AAV antibodies might severely affect transduction efficiency after intrathecal injections. Additionally, the authors

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reported a similar distribution pattern for both delivery routes, with greater astrocytic than neuronal tropism in adult animals. In conclusion, it is not completely clear if IT

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administration circumvents the possible limitations of intravascular injections. Furthermore, IT delivery requires a more invasive and painful procedure than a simple

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IV injection, which is normally considered the ideal delivery system for clinical trials.

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Conclusion

AAV vectors emerged as the preferential platform for CNS gene therapy, due to their numerous favorable characteristics. Beyond all the available serotypes and

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delivery routes, systemic AAV9 administration is considered a highly promising approach for clinical applications. In fact, it provides CNS widespread transgene expression through a simple and minimally invasive procedure. This is possible due to AAV9 ability to cross the BBB, which has already been confirmed in rodents, cats, dogs and non-human primates. Nevertheless, its preferential transduction profiles in animal models at different ages remains to be elucidated, requiring further investigation. According to previous studies, the best candidates for this therapy would be disorders characterized by: i) involvement of spinal cord motor neurons, ii) aberrant glial function and iii) early-onset neuronal pathology in the brain. Newborn AAV9 administration might be an advantageous option in easily diagnosed disorders, since NAbs in children are lower than in adults and early intervention requires less viral particles. On the other hand, treatments during adulthood might require optimization methods to enhance neuronal transduction. 31

ACCEPTED MANUSCRIPT Several preclinical reports have already shown successful results for a wide range of CNS disorders, leading to the first clinical trial. This is an extremely important step in the field because it will reveal whether the remarkable results obtained in

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animal models are reproduced in humans.

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In conclusion, AAV-mediated gene therapy has recently produced some

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remarkable results in clinical trials. The recent technological leap with the introduction of vascular infusion of AAV9 to reach widespread gene delivery to the CNS raises the

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expectation of further successes in gene therapy for neurological disorders.

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Acknowledgements

The authors thank M. Sena-Esteves (University of Massachusetts Medical School, USA) for manuscript review.

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Our group is supported by funds FEDER and the Competitive Factors Operational Program – COMPETE and by national funds through the Portuguese Foundation for Science and Technology (PTDC/SAU-NMC/116512/2010, E-Rare4/0003/2012, PTDC/NEU-NMC/0084/2014 to LPA, EXPL/NEU-NMC/0331/2012, SFRH/BPD/66705/2009 to RJN); by the JPND cofunding JPCOFUND/0001 and 0005/2015 (LPA), by the Richard Chin and Lily Lock Machado Joseph Disease Research Fund (LPA); and the National Ataxia Foundation (LPA and RJN).

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