Engineering hematopoietic stem cells toward a functional cure of human immunodeficiency virus infection

Cytotherapy, 2016; 18: 1370–1381

Engineering hematopoietic stem cells toward a functional cure of human immunodeficiency virus infection

JIANBIN WANG & MICHAEL C. HOLMES Sangamo BioSciences Inc., Richmond, California, USA Abstract The battle with human immunodeficiency virus (HIV) has been ongoing for more than 30 years, and although progress has been made, there are still significant challenges remaining. A few unique features render HIV to be one of the toughest viruses to conquer in the modern medicine era, such as the ability to target the host immune system, persist by integrating into the host genome and adapt to a hostile environment such as a single anti-HIV medication by continuously evolving. The finding of combination anti-retroviral therapy (cART) about 2 decades ago has transformed the treatment options for HIV-infected patients and significantly improved patient outcomes. However, finding an HIV cure has proven to be extremely challenging with the only known exception being the so-called “Berlin patient,” whose immune system was replaced by stem cell transplants from a donor missing one of HIV’s key co-receptors (CCR5). The broad application of this approach is limited by the requirement of an HLA-matched donor who is also homozygous for the rare CCR5 delta32 deletion. On the other hand, the Berlin patient provided the proof of concept of a potential cure for HIV using HIV-resistant hematopoietic stem cells (HSCs), revitalizing the hope to find an HIV cure that is broadly applicable. Here we will review strategies and recent attempts to engineer HIV-resistant HSCs as a path to an HIV cure. Key Words: gene therapy, human immunodeficiency virus–resistant, stem cells, transplantation

Introduction

Viral reservoirs and a functional cure

Human immunodeficiency virus (HIV) is the causative agent of acquired immune deficiency syndrome (AIDS). With 36.9 million people infected worldwide, HIV infection stands as a huge health and economic burden, especially in sub-saharan Africa. The development of highly active anti-retroviral therapy (HAART) or combination antiretroviral therapy (cART) initiated about 2 decades ago has transformed the treatment options for HIV-infected patients and significantly improved patient outcomes [1,2]. However, cART can neither eliminate latent reservoirs of virus nor fully restore the health of patients [3–5]. Successful control of HIV infection requires a lifetime of excellent patient adherence to the treatment protocol, and the medications themselves can be quite expensive. Furthermore, the pharmacological toxicities associated with the treatment regimen can be intolerable for some patients. Hence, searching for an HIV cure is the ultimate goal of many researchers. With the recent report of the first cured Berlin patient [6,7], the field is revitalized with the hope of finding a broadly applicable cure.

HIV is an enveloped RNA virus. HIV infection is initiated through the binding of viral envelop protein gp120 to cellular CD4 receptor, followed by conformational changes in gp120 and subsequent binding to the CCR5 or CXCR4 co-receptor, which then triggers structural changes in gp41 and ultimately leads to membrane fusion and virus entry. Viral genomic RNA (gRNA) is released into the cytoplasm upon uncoating and is reverse transcribed into viral genomic DNA (gDNA). Viral gDNA is translocated into the nucleus of the infected cell in the form of a preintegration complex, and then integrated into host chromosomes (provirus). After transcription and translation, viral proteins and gRNA are packaged (assembled) into a new virion and released from the cell membrane by budding to spread the infection [8]. Various processes in the HIV life cycle are targeted for anti-HIV therapy [9]. Most components of the current cART regimen mainly target the post-entry process and are highly efficient in inhibiting HIV replication. However, once the virus gets integrated into the host cell genome and becomes dormant, it is mechanistically difficult to remove the integrated viral DNA.

Correspondence: Jianbin Wang, PhD, Sangamo Biosciences, Inc., 501 Canal Blvd, Richmond, CA 94804, USA. E-mail: [email protected] or [email protected] (Received 16 March 2016; accepted 21 July 2016) ISSN 1465-3249 Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2016.07.007

HIV-resistant HSCT for a functional cure The infected cells, especially those long-lived cells such as memory T cells, with dormant virus form a major component of the viral reservoirs [10,11]. Based on the definition by amfAR, the Foundation for AIDS Research, to be considered cured, an infected person would need to meet three criteria: (1) be able to live a normal, healthy lifespan; (2) be off antiretroviral therapy or any other HIV-related medications; and (3) be incapable of transmitting the virus to others. A “sterilizing” cure, comprising a complete eradication of all replication-competent HIV from the body, is desirable but would be extremely difficult to achieve and nearly impossible to prove [12,13]. Thus, a functional cure, where the virus may not have been completely eliminated but would be effectively controlled and prevented from causing disease progression may be the most realistic goal in the near term [13]. A functional cure generally means people can live a reasonably normal life, perhaps a completely normal life, without the need for cART. Remission or long-term remission, the latest definition to enter the HIV cure dialog, refers to control of HIV viral load at low levels (such as <50 copies/mL) in the absence of cART treatment for a relatively long period of time (unspecified so far), which could have the potential to rebound due to low levels of HIV replication [14]. T.B., the Berlin patient, represents a functional HIV cure, and possibly a sterilizing cure [6]. He had both HIV and leukemia and underwent intensive radiation and chemotherapy, which can potentially wipe out a large portion of viral reservoirs, and received hematopoietic stem cell transplantation (HSCT) twice from an HIV-resistant donor with the “delta 32” mutation in the CCR5 gene (CCR5 Δ32/Δ32).The 32-base pair (bp) deletion (Δ32) in the CCR5 gene causes a frame shift mutation at position 185 and in turn leads to dysfunction and diminished surface expression of the CCR5 receptor. Individuals with a homozygous Δ32 deletion are consequently highly protected from infection with HIV-1.The naturally occurring mutation is most common in white populations where about 1% of the population are homozygous for the mutation [15,16]. The most ultrasensitive tests have detected no replication-competent HIV in the patient’s tissues collected about 5 years after his radical treatment. In addition, anti-HIV antibody and cytotoxic T lymphocyte (CTL) responses decreased or disappeared [7].The Berlin patient has remained off of cART for about 9 years now. Of note, the stem cell donor was both an appropriate tissue match and homozygous for the CCR5Δ32 mutation, which is extremely rare. In addition, a graft-versus-host disease (GVHD) developed after the treatment, where donor-derived immune cells likely attacked/eradicated residual leukemia cells as well as HIV-infected donor immune cells including viral reservoirs. More recent studies of the two “Boston patients”

1371

[17,18], who also received cytoablative conditioning and allogenic HSCT, but from wild-type CCR5 donors, initially reported markedly reduced viral reservoirs and undetectable HIV viremia for months. However, the virus later returned in both patients after ceasing cART, highlighting the potential necessity of using HIVresistance HSCs as donor cells for transplantation to attain a cure. The “Mississippi baby” and the 14 post-treatment controllers from the ANRS VISCONTI study (ViroImmunological Sustained CONtrol after Treatment Interruption study funded by the French National Agency for Research on AIDS and Viral Hepatitis) [19–21], who had anti-HIV treatment initiated very early and had remission without viremia for much longer than the typical 2–4 weeks after therapeutic interruption, provided evidence for the effective control of viral reservoirs by starting cART early after infection. Early treatment is thought to potentially reduce the size of viral reservoirs, preserve patients’ immune responses and protect them from chronic immune activation. However, despite the early hope of a cure for this child, detectable HIV has now been measured in the Mississippi baby’s blood.Thus, these studies highlight how stealthy and pernicious HIV is and how challenging it can be to eradicate viral reservoirs with cART alone. Multiple strategies have been pursued to eradicate the viral reservoirs. One of the most discussed and tested strategies is the so called “shock and kill” or “kick and kill.”The dormant reservoirs are activated by various latency reversing agents (LRAs) and subsequently eliminated by the viral cytopathic effect or by the immune system in the presence of cART to prevent new infections [22,23]. Several epigenetic and non-epigenetic LRAs have been tested and appear promising, including inhibitors of histone deacetylases (HDAC), histone methyltransferase (HMT), and DNA methyltransferases (DNMT), protein kinase C (PKC) agonists and others [22,23]. However, portions of the HIV reservoir (the “deep reservoir”) appear to be refractory to LRAmediated activation [24]. Alternatively, the LRAs tested have poor in vivo pharmacokinetic activity to reach and activate the target cells, or possibly not all infected cells were eliminated by the viral cytopathic effect or the immune system following LRA-mediated activation [25]. Hence, several immunologic strategies (immunotherapy) are being explored to augment the capacity of the host immune system to eliminate viral reservoirs, including therapeutic vaccines, broadly neutralizing monoclonal antibodies, HIV-specific CTLs and various immune modulating agents including those that target the immune checkpoints PD-1 and CTLA-4 [22,26,27]. However, the ability of HIV-1 to destroy activated CD4 T cells and the defect in normal CD4 T-cell function in HIV-infected patients have probably played an important role in the largely unsuccessful attempts so far.

1372

J.Wang & M. C. Holmes

The role of CD4+ T-cell depletion and immune dysfunction in HIV pathogenesis: the potential role of HIV-resistant cells might play during an HIV cure CD4+ helper T cells orchestrate both cellular and humoral immune responses. The depletion and functional defects of CD4+ T cells play a critical role in HIV pathogenesis [28,29]. HIV-associated chronic immune activation is likely the major driving force of disease progression by stimulating viral replication, which leads to a gradual decrease in the number of CD4+ T cells. CD4+ T-cell depletion is linked to direct viral cytopathic effect and bystander effects of infected or activated cells, immune activation and immune senescence (aging of the immune system) [30]. In fact, the most important damage to T-cell homoeostasis happens very early in the gastrointestinal tract with a massive depletion of activated CCR5+ CD4+ T cells [31]. In addition to the loss of CD4+ T cells in quantity, a profound loss in T-cell quality also occurs, including preferential loss ofT-helper-17 (Th17) cells and mucosal-associated invariant T cells, which are an important defense against bacteria [32,33]. CD4+ T-cell help is critical for the generation of effective and persistent CD8+ CTL responses. CD4+ T cells interact with CD8+ T cells and have the ability to produce interleukin (IL)-2 or other cytokines to promote CD8+ T-cell proliferation. Defective CD4+ T-cell help can lead to the poor quality of CD8+ T-cell responses [28,29]. In addition, CD4+ T-cell help is also important for B-cell maturation and proliferation. For example, abnormality in follicular CD4+ T-helper (Tfh) cells is a driving force for hyperglobinopathy in patients with HIV [28,29]. Even though most patients with HIV have a substantial amount of CD4+ T cells by continuous replenishment, the selective depletion of activated T cells leads to an insufficient amount of HIV-targeting cells, including the functionally most important ones. Therefore, introduction of HIVresistant CD4+ T cells, which is the missing piece of anti-HIV immunity, by infusing either genetically modified T cells or HSCs is one of the most promising therapeutic approaches with the greatest potential to reconstitute a patient’s immune system. Strategies to generate HIV-resistant cells HIV-resistant cells can be generated by targeting different stages of the viral life cycle.This can be achieved by targeting either viral gene/RNA/protein or host cell receptors/factors, for example, CCR5 [34,35], CXCR4 [36,37] and gp41 [38–40] for viral entry; alternated TRIM5α for uncoating [41]; lens epithelium-derived growth factor (LEDGF)/p75 for integration [42]; tat/ transactivating region (TAR) for transcription [43,44]; rev/RRE for RNA export [45,46]; gag, tumor suscep-

tibility gene 101 (TSG101) and Tetherin/BST-2 for viral assembly and release [47–51]. However, targeting the pre-integration steps is favored over targeting the post-integration steps because the later has the potential to promote the survival of infected cells. Multiple approaches targeting different stages of the HIV life cycle can be used to generate cells either resistant to HIV infection or unable to support HIV replication and spreading. The majority of the approaches focus on antagonizing or knocking-down viral or host genes, such as RNA-based methods, including anti-sense RNA (asRNA), small or short interfering RNA (siRNA), small or short hairpin RNA (shRNA), RNA aptamers, decoys, and ribozymes [52], and transgene-based methods, such as expression of dominant negative inhibitors, intrabodies, intrakines, fusion inhibitors and altered TRIM5α [53]. Another way to generate HIV-resistant cells is to permanently knock-out host genes essential for HIV infection using targeted nucleases [54]. A general comparison of different strategies to generate HIV-resistant cells is summarized in Table I. Anti-sense RNAs are single-stranded RNA molecules that are complementary to the target messenger RNA (mRNA) and can inhibit translation by base pairing to the intended target and physically obstructing the translation machinery or inducing degradation. Strategies to target either viral or host genes using asRNAs are being explored [44,55].The clinically most advanced asRNA is VRX496 delivered by a “conditionally replicating” lentivirial vector, where the RNA targets the HIV env region of viral transcripts.The adoptive transfer of CD4+ T cells genetically modified by VRX496 was demonstrated to be safe in HIV-infected subjects with a modest immunologic benefit [56]. Nucleic acid aptamers are oligonucleotides (RNA or DNA) that are selected from a large library for binding to a specific target RNA, DNA or protein. A number of aptamers have been developed against various HIV-1 viral genes or elements [57]. RNA decoys can be considered as a special class of aptamers. RNA decoys are single-stranded RNA molecules that are usually identical to or have a similar structure or binding capability as the target RNA/DNA, and can be used to competitively block the function of the target RNA/DNA. HIV rev response element (RRE) and the TAR are the most explored targets, where aptamers against these targets are included as components in some of the complex anti-HIV constructs being developed [58–60]. siRNAs are 20–25 bp long, double-stranded RNA molecules that can activate the RNA interference (RNAi) pathway to degrade the target mRNA resulting in reduced or diminished translation. A shRNA is the precursor of siRNA and can be converted to siRNA by Dicer inside a target cell. SiRNA/shRNA have been exploited to target various viral or cellular

HIV-resistant HSCT for a functional cure

1373

Table I. Strategies to generate HIV-resistant cells.

Strategies Knock-down strategy RNA-based technologies Anti-sense RNA Aptamer and RNA decoy Ribozyme shRNA/siRNA

Transgene-based technologies Dominant negative inhibitor Intrabody and intrakine Altered TRIM5a Fusion inhibitors Knock-out strategy Targeted nucleases (ZFN, TALEN, Meganuclease, CRISPR/Cas9)

Typical viral targets

Typical host targets

Pros

Cons

RRE, TAR, tat RRE, TAR, env Most viral genes Entire viral genome

CCR5, CXCR4 CCR5, CD4 CCR5, CD4 CCR5, CXCR4, CD4

(1) Anti-sense and sh/ siRNA are easy to design; (2) Can be multiplexed or combined

(1) Inhibition might be incomplete; (2) Require persistent expression; (3) For viral targets: viral escape mutants; (4) For host targets: may affect physiological function

Rev N/A N/A

Fusion inhibitors are highly potent

(1) Require persistent expression; (2) For host targets: may affect physiological function

gp41

N/A CCR5, CXCR4 TRIM5a/ Cyclophilin N/A

LTR

CCR5/CXCR4

(1) Complete knockout co-receptor; (2) Transient expression is sufficient

For host targets: (1) Single KO is not sufficeint for dual tropic virus; (2) May affect physiological function

ZFN, zinc-finger nucleases; TALEN, transcription activator-like effector nuclease; CRISPR, clustered regularly interspaced short palindromic repeat; Cas9, CRISPR associated protein 9; RRE, rev response element; N/A, not applicable; LTR, long terminal repeat; KO, knock-out.

targets and are components of therapeutic agents being examined in a handful of clinical trials [60–62]. Ribozymes (ribonucleic acid enzymes) are a special class of RNA molecules that possess protein enzymelike activity and are capable of catalyzing specific biochemical reactions [63]. Ribozymes can be engineered and used to cleave target mRNA to reduce or diminish its translation. Ribozymes have been exploited to target various viral or cellular targets and are also being evaluated in early-stage clinical trials [60,64–66]. Dominant negative inhibitors can be considered as protein decoys or aptamers that competitively bind the partners of the target protein but do not transduce the proper signal downstream, resulting in reduced or diminished function of the target protein. A transdominant-negative rev has already been tested in a clinical trial [67]. Intrabodies and intrakines can also be considered as special protein aptamers. Intrabodies are antibodies produced inside of a cell that are able to bind to the target protein intracellularly and prevent it from being expressed on the cell surface. Intrakines are cytokines produced inside of a cell that are able to bind to the target receptor intracellularly and prevent it from being expressed on the cell surface. Intrabodies and intrakines against CCR5 or CXCR4 have been explored for their potential as anti-HIV agents [68–70]. A membrane anchored fusion inhibitor maC46, which is mainly derived from the C-heptad repeat region of gp41 and can block the formation of the six

bundle structure during virus-cell fusion, is another promising anti-HIV agent [71]. It has been shown experimentally to be superior to an HIV-1 tat/revspecific shRNA and an env-specific asRNA [39]. Cells expressing maC46 showed a significant survival advantage upon HIV challenge in vitro and in vivo [39,40,71]. A phase 1 clinical trial was conducted where autologous T cells transduced with maC46 were infused into patients. The approach is confirmed to be safe. However, most likely due to insufficient levels of the introduced gene-protected T cells (<0.0001– 0.8% of total peripheral blood leukocytes), no clear anti-viral effect was observed [38]. Hematopoietic stem and progenitor cells (HSPCs) transduced to express maC46 were recently tested in an autologous transplantation model in non-human primates (pigtail macaques). Following simian/human immunodeficiency virus (SHIV) challenge, maC46-treated but not control macaques showed a positive selection of genemodified CD4 + T cells in the peripheral blood, gastrointestinal tract and lymph nodes. In addition, maC46-treated macaques also maintained high frequencies of SHIV-specific, gene-modified CD4+ T cells, an increase in non-modified CD4+ T cells, enhanced cytotoxic T lymphocyte function and antibody responses [40]. The results suggest that an autologous transplantation of HIV-resistant HSPCs has the potential to reconstitute anti-HIV immunity. To bring fusion inhibitors like maC46 to the exact site of HIV-target cell interaction, together with our collaborators we designed and validated C34-coreceptor

1374

J.Wang & M. C. Holmes

fusion proteins for inhibition of HIV infection. Similar to C46, C34 is a short peptide derived from the C-heptad repeat region of gp41. Primary CD4+ T cells expressing C34-coreceptor are protected from both CCR5- and CXCR4-tropic HIV-1 infection, suggesting that this represents a novel method to generate HIV-resistant cells [72]. Other knock-down techniques such as DNAzyme and peptide nucleic acid (PNAs) have also been or are being explored for their potential application in anti-HIV therapy. DNAzymes are engineered DNA molecules with catalytic activity. Similar to ribozymes, DNAzymes can be exploited to target/cleave HIV tat/ rev transcript to reduce/diminish their expression [73]. PNAs are DNA mimics in which sugar phosphate backbone is replaced with N-(2-amino ethyl) glycine units. This modification makes them highly stable in the cellular compartment. Anti-sense PNA targeted against different conserved regions of the HIV genome are being developed. However, multiple PNAs targeting different conserved regions of the viral genome are required to be used in combination to reach maximum inhibition of viral replication [74]. Instead of antagonizing or knocking-down viral or host genes, which can be transient or incomplete, another promising anti-HIV strategy is to permanently knock-out host genes essential for HIV infection by targeted nucleases to generate HIV-resistant cells. Programmable targeted nucleases, which include zincfinger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)/Cas endonucleases, are a powerful class of enzymes that promote genome editing through the creation of a site-specific DNA double-strand break (DSB) at a pre-defined site in the genome [75]. Repair of DSBs can proceed via the non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways [76,77]. NHEJ is usually more efficient in mammalian cells and can be used mainly for gene disruption applications. HDR uses a homologous donor sequence as a template for the conservative repair of the DNA break and can be used for gene correction or gene addition, in addition to gene disruption. The major advantages of using targeted nucleases are as follows: (1) only transient treatment is required; (2) the genetic modification is permanent for the life time of the cell and is inheritable; (3) for transgenes or corrected genes, gene expression can be controlled by an endogenous promoter to achieve appropriately regulated and physiological levels of expression; and (4) for transgenes, the integration is site-specific, not random, providing a potentially more uniform level of expression and better safety profile than other methods. Under the context of gene modified HSCs, these features will be even more important for the safety

and efficacy of the modified cell product. Recent advances in achieving significantly improved efficiencies of targeted integration in HSPCs will allow broad application of different genome editing strategies for HIV, including the simultaneous disruption and addition of genes targeting the HIV life cycle [78]. ZFNs are engineered endonucleases that contain two linked domains, a DNA-binding domain of a versatile class of eukaryotic transcription factors, zinc finger proteins (ZFPs), and the nuclease domain of the FokI restriction enzyme. ZFPs are generated by combination of modular design of single or multimers of individual zinc fingers, which can each recognize three bases in an array of genomic DNA sequence (usually 18–36 bp total length for a pair of ZFNs) [79]. Similar to ZFN, TALENs are a fusion of the transcription activator-like effector (TALE) DNA binding domain and the FokI nuclease domain. TALEs are generated by combination of multiple repeats of single modules with repeat-variable di-residues (RVDs), which each recognize a single base of genomic DNA sequence [80]. Different from both ZFNs and TALENs, the CRISPR/ Cas system recognizes its target DNA based on RNADNA base pairing instead of protein-DNA interactions. Due to the relative ease of constructing guide RNAs for a given target, the CRISPR/Cas9 technology has been widely adapted by the research community. Researchers are actively working on ways to improve the relatively lower target-specificity of the system to use it for possible clinical applications [81]. Among the various potential targets, CCR5 is the most-studied knock-out target for generation of HIVresistant cells.This approach is also the most advanced in that it is currently being evaluated in early-stage clinical trials testing genome-edited autologous T cells or edited HSPCs. Besides ZFN-mediated disruption of the CCR5 gene [34,35,82], both TALEN and CRISPR/Cas9 have also been designed and tested to disrupt the CCR5 gene [83–85]. Although CCR5 knock-out is a promising anti-HIV approach, it has also raised some concerns. One concern is that CCR5 knock-out may prevent the migration of T cells and monocytes/macrophages in response to its natural chemokine ligands. Studies using CCR5 knock-out mice demonstrated that CCR5 knock-out can be either detrimental or beneficial to the host depending on the specific types of pathogens tested [86–92], suggesting a complex interplay between the immune system and the pathogen. Studies involving CCR5 Δ32 homozygous individuals suggested that the CCR5 Δ32 mutations can be beneficial to the host in some infections (e.g., HIV-1, Staphylococcus aureus, possibly smallpox and Yersinia pestis) [93–96], but detrimental in others (e.g., tick-borne encephalitis and West Nile virus) [97,98]. The fact that there are largely unnoticeable differences between CCR5 Δ32 homozygous

HIV-resistant HSCT for a functional cure individuals and the rest of the population suggests that CCR5 can be safely targeted for an HIV cure. In addition, the unmodified portion of the immune system is expected to provide at least part of the physiological CCR5 functions. Another concern is that CCR5 knock-out alone is not protective against CXCR4tropic HIV-1 strains. For this consideration, only patients with CCR5-tropic HIV are enrolled in the clinical trials using CCR5 knock-out cells. To overcome the limitation of CCR5 knock-out, targeting of CXCR4 or simultaneous targeting of both CCR5 and CXCR4 are also being pursued where studies have demonstrated that the knock-out cells are resistant to CXCR4 or both CCR5 and CXCR4-tropic viruses, respectively, providing survival advantages in animal models [36,37,99]. CXCR4 knock-out is lethal to embryo development in the mouse, suggesting an important physiological function of CXCR4 during early development [100]. However, knock-out of CXCR4 in adult cells is most likely tolerable. In fact, a CXCR4 antagonist, plerixafor, is widely used for HSC mobilization without significant adverse effects [101]. HSC for an HIV cure Mature blood and immune cells have a finite life span and must be continuously replaced throughout life. The stem cells that form blood and immune cells are known as HSCs, which are ultimately responsible for the constant renewal of blood cells via the production of billions of new blood and immune cells each day [102,103]. HSCs are multipotent, self-renewing progenitor cells that develop from mesodermal hemangioblast cells. A single primitive HSC has the capability to generate the entire hematopietic system as demonstrated in animal engraftment studies. HSCs can be found in adult bone marrow, peripheral blood and umbilical cord blood, and can be harvested for treating many diseases. HSCTs are now routinely used to treat patients with cancers and other disorders of the blood and immune systems. HSCs can potentially be used for the treatment of an even wider variety of diseases, especially considering the recent findings that HSCs can probably transdifferentiate and generate other types of cells, such as muscle, blood vessels and bone [102,103]. HSCs are routinely enriched based on expression of the cell surface marker CD34 [104]. However, CD34+ cells are a heterogeneous population, with the majority of cells being committed progenitors, named hematopoietic progenitor cells (HPCs), which are restricted to certain lineages lacking the ability to selfrenew. Hence, purified CD34+ cells are often referred as HSPCs but not HSCs. Bone marrow used to be the main source of HSCTs, but has largely been replaced by mobilized peripheral blood HSPCs now. In

1375

addition, umbilical cord blood CD34+ cells are also increasingly being used, especially for small patient recipients [105]. Allogeneic HSCT, limited by histocompatibility, is usually not considered for treating HIV unless it is indicated for an additional disease condition such as cancer, due to the inherent significant morbidity and mortality associated with the treatment procedure and the possibility of development of GVHD following transplantation [106]. On the other hand, manageable GVHD may be useful in helping to purge the HIV reservoirs. An HIV cure is possible if allogeneic HSCT is conducted using HIV-resistant stem cells, as demonstrated in the Berlin patient [6]. With the recent advances in transplantation technology, haploidentical HSCT is also becoming a feasible approach [107]. Autologous transplants, which eliminate the risk for patients to develop GVHD, have the potential for being broadly applied to HIV patients. Autologous T-cell transplantation for treating HIV disease is currently being tested in clinical trials [56,108]. Autologous HSCT may provide several advantages over adoptive T-cell therapy, such as (1) besides HIV-resistant T cells, other HIV-resistant hematopoietic lineages can also be derived from the transplanted HSC to replace part of the viral reservoirs such as tissue macrophages; (2) more cells with long-term potential will be engrafted, which can ensure possibly higher levels of HIVresistant cells for longer duration; and (3) the possibility to reconstitute a new and wholly HIV-resistant immune system de novo in the patient. A path to reach a sterilizing cure will likely need to use a myeloablative regimen to remove most of the old viral reservoirs and create space for an allogeneic graft comprising gene-modified allogeneic HSPCs that are selected/purified to be 100% HIV-resistant cells, an allogeneic HSCT to reconstitute an HIVresistant immune system and to eradicate any remaining reservoirs by GVHD (100% chimerism). This path is currently considered highly risky, such that only a limited number of the HIV patient population who have accompanying malignancies and HLA matched donors can be candidates for such an approach (Figure 1A). A more broadly applicable approach will be nonmyeloablative or reducedintensity conditioning regimen that will remove only part of the viral reservoirs and create space for engraftment, which again will comprise relatively high levels of gene-modified autologous HSPCs with or without ex vivo selection, autologous HSCT to reconstitute a mixed immune system with a significant portion of the cells being HIV-resistant, in vivo chemoselection or treatment interruption, can also be applied if needed. Eventually, with the help of the HIVresistant cells, especially HIV-resistant CD4+ T cells, a functional immune system can be established and

1376

J.Wang & M. C. Holmes

A * * ** **

HSPCs

+ +

+

+ + *

Anti-HIV agent HIV-resistant cell HIV reservoir

+

Sterilizing cure

Myeloablative conditioning

+

Modified HSPCs

* +

Selection

+ +

+

Allogeneic

Immune reconstitution

transplantation

Chronic GVHD

+ +

100% modified HSPCs

B * * ** **

HSPCs

+ +

+

+ Non-myeloablative conditioning

+

Modified HSPCs

Functional cure *

*

Enrichment

+

(optional)

+

+

* Autologous

+

transplantation

*

Immune reconstitution

+ +

In vivo selection (optional)

Enriched HSPCs Figure 1. A roadmap to a sterilizing cure by allogeneic HSCT or a functional cure by autologous HSCT.

HIV can be controlled in the absence of cART (functional cure, Figure 1B). It is currently unclear what percentage of HIV-resistant HSCs are required to reconstitute such a mixed immune system that has the ability to control or eradicate HIV infection. Clinical trials testing gene-modified HSCT in HIV-infected patients Gene therapy using modified HIV-resistant HSPCs has the potential to reconstitute an HIV-resistant

immune system. A few HSCT-based gene therapy approaches have already been tested in clinical trials (Table II), including a RRE decoy [109], ribozymes targeting tat, tat/rev or tat/vpr combinations [110–113], a transdominant rev (TdRev) [67,114,115] or triple combinations of tat/rev-targeting shRNAs, a RRE decoy and a CCR5-targeting ribozyme [60]. Retroviral vectors were used for delivery in all these studies except the most recent one using the triple combinations, which were delivered with a lentiviral vector [60]. Low levels of in vivo gene marking appear to be a major

HIV-resistant HSCT for a functional cure

1377

Table II. Clinical trials testing gene-modified HSCT in HIV-infected patients. Status Completed Completed Completed Completed Completed Completed Completed Suspendedb Ongoing Ongoing Ongoing

Testing agents

Delivery

HSPC source

References

RRE decoy RRz2 (tat ribozyme) rev/tat ribozyme huM10 (transdominant Rev) TdRev (trans-dominant Rev) OZ1 (tat/vpr ribozyme) rHIV7-shI-TAR-CCR5RZa M87o (maC46) LVsh5 (CCR5 shRNA)/C46 C46/CCR5 (ribozyme)/P140K SB-728mR (CCR5 ZFN)

Retrovirus Retrovirus Retrovirus Retrovirus Retrovirus Retrovirus Lentivirus Retrovirus Lenti virus Lenti virus mRNA

Autologous, BM Autologous, mPB Autologous, BM Autologous, BM Allogeneic, mPB Autologous, mPB Autologous, mPB Autologous, mPB Autologous, mPB Autologous, mPB Autologous, mPB

Kohn et al. 1999 Amada et al. 1999, 2004 Michienzi et al. 2003 Podsakoff et al. 2005 Kang 2002; Hayakawa 2009 Mitsuyasu et al. 2009 Digiusto et al. 2010 NCT00858793 NCT01734850 NCT02343666 NCT02500849

BM, bone marrow; mPB, mobilized peripheral blood. a A triple combination of tat/rev shRNAs, a TAR decoy and a CCR5 ribozyme. b A leukemia case was reported in a patient treated with a similar retrovirus vector. For safety reasons, the investigator voluntarily stopped recruitment for this trial.

challenge, with most studies having either undetectable or very low levels (about 0.01%) of gene marking at 6 months or 1 year after HSCT. Autologous HSCT was used for most of these clinical trials except one, in which allogeneic HSCT was used and 100% chimerism was achieved, but still only 0.01% gene marking was observed [67,114]. Possible reasons for such low levels of gene marking may include an inadequate frequency of modified long-term HSCs. With regard to the transduction efficiency, about 10%-35% of total cells were transduced using retroviral vectors. However, it is likely that the real transduction efficiency for quiescent HSCs is markedly lower. With regard to transduction frequency of the most primitive HSCs, the inclusion of the stimulatory cytokine IL-3 in the culture medium is a concern for the trials using retroviral vectors because transducing the cells with IL-3 probably reduced the overall frequency of the most primitive HSCs significantly. The recent development of safer and more efficient lentiviral vectors has the potential to alleviate these problems. However, the presence of anti-HIV medicine in cART-treated HSPCs can partly counteract such an advance [116]. Further optimization or the choice of an alternative delivery system can be beneficial. Another possible cause of poor gene marking may be from low HSPC engraftment.This is likely a problem for autologous HSCT, especially for the grafts using no pre-conditioning.The choice of an optimal non-myeloablative conditioning regimen can likely make a significant difference. Clearance of genemodified cells by host immune system may also be an issue. This possibility has not been fully ruled out so far, but at least it does not appear to be the main cause, especially in the setting of allogeneic HSCT where 100% chimerism was established within a year. Finally, survival or growth disadvantage of gene-modified cells may cause low marking, however, this is unlikely the cause. In fact, a survival advantage of modified cells

was observed in the time period of active HIV replication when anti-HIV treatment was interrupted [109]. However, the transient increase of gene-modified cells also suggested that short-lived CD4+ T cells were selectively expanded but not HSCs because there is no direct selective pressure on HSPCs from HIV infection. There are also a few ongoing HSCT trials (Table I), testing the following: (1) maC46 (ClinicalTrials.gov #NCT00858793); (2) a combination of the maC46 and a CCR5-targeting shRNA (#NCT01734850); (3) a combination of the maC46, a CCR5-targeting ribozyme, and an in vivo selection marker, the O6methylguanine DNA methyltransferase (MGMT) P140K mutant (#NCT02343666); and (4) CCR5targeting ZFNs (#NCT02500849). It is still an open question whether these novel approaches can reach or bring us much closer to a functional cure. However, at minimum, results of these trials will help us to better understand ways to reconstitute an anti-HIV immune system in the presence of chronic HIV infection. Challenges and hopes With regard to finding an HIV cure, a major challenge faced with both allogenic and autologous HSCT is the efficiency of gene modification of the transplantable donor cells, which will have a significantly lower efficiency of HIV-resistant cells than what were used for the Berlin patient (100% CCR5 Δ32/Δ32 HPSCs). Furthermore, the frequency of HIV-resistant cells will get even lower after transplantation, especially in case of autologous HSCT with reduced intensity conditioning. The presence of a substantial amount of HIV-susceptible cells in the donor cells, which can be potentially exploited by HIV to generate new viral reservoirs, is a great concern for an HIV cure strategy. Unless cART is 100% efficient in preventing this to occur, or alternatively, the reconstituted

1378

J.Wang & M. C. Holmes

immune system is able to control or eradicate HIV infection, the residual susceptible cells could serve to facilitate HIV replication.Toxicity associated with chemotherapy pre-conditioning before HSCT can frequently cause the necessity to pause cART, at least temporarily, suggesting that we cannot always rely on cART unless a more tolerable regimen is available. Hence, eventually we may have to rely on the reconstituted mixed immune system to control or eradicate HIV. Numerous studies have highlighted the difficulty in controlling HIV infection by a normal HIVsusceptible immune system. However, it is currently unknown whether an HIV-resistant immune system can generate efficient immune responses to control or eradicate HIV-infected cells when co-existing with HIVsusceptible cells. Animal studies suggested that HIVresistant cells have a survival advantage in the presence of HIV challenge [34–37,99]. In addition, HIVresistant cells appear to be able to establish antiHIV immunity in a non-human primate model [40]. Importantly, the anti-HIV immunity has also helped the survival of HIV-susceptible cells [40].These results suggest that with the initial help of cART and the presence of HIV-resistant CD4+ T cells, anti-HIV immunity can be developed and maintained. The hope is that such anti-HIV immunity can eventually keep HIV infection under control even in the absence of cART. In case that HSCT of HIV-resistant cells is not sufficient by itself to cure HIV, it can be combined with an in vivo chemo-selection method, such as the MGMT P140K selection approach that is independent of the cytopathic effect of HIV replication and has the potential to reduce viral reservoirs by itself [117]. This can potentially be a path to reach a complete eradication of HIV reservoirs and a sterile cure. Alternatively, combination of HIV-resistant HSCT with immunotherapy approaches such as engineered CTLs or therapeutic vaccines may also hold promise for a sterilizing or functional HIV cure. We have recently found an efficient way to genome edit HSPCs by homology-directed repair, by which we can simultaneously disrupt a gene such as CCR5 and insert a transgene such as a selection marker gene or an expression cassette to augment the protection or antiHIV activity of the engrafted cells [78].With the recent advances in technologies to genetically modify hematopoietic cells, we believe finding a broadly applicable HIV cure is challenging but reachable in the foreseeable future. Concluding remarks HIV is one of the most sophisticated and the most difficult viruses to conquer. With more than 30 years of ongoing work to develop an effective treatment for HIV, numerous advances have been achieved. This once

deadly disease is now managed by adapting the regimen of cART. However, HIV’s ability to destroy and evade the immune system and the persistence of viral reservoirs has prevented its clearance from infected patients.The Berlin patient revitalized our hope to cure HIV. Our ability to generate HIV-resistant cells including HSCs at clinical scale may hold the key to reconstitute an HIV-resistant immune system, which can potentially lead to immunologic control or eradication of HIV. HSCT using HIV-resistant cells can also be combined with in vivo chemo-selection approaches or anti-HIV immunotherapy to possibly achieve a sterilizing or functional cure. Acknowledgments We thank Paula L. Cannon (University of Southern California), David L. DiGiusto and John A. Zaia (City of Hope) for their collaborative work, the California Institute for Regenerative Medicine (CIRM; DR101490 and RT3-07848) and the National Institute of Allergy and Infectious Diseases (NIAID; U19 AI117950) for their support and Susan Abrahamson (Sangamo) for critically reading the manuscript and helpful suggestions. Disclosure of interests: All authors are full-time employees of Sangamo BioSciences, Inc. References [1] Gulick RM, Mellors JW, Havlir D, Eron JJ, Gonzalez C, McMahon D, et al. Treatment with indinavir, zidovudine, and lamivudine in adults with human immunodeficiency virus infection and prior antiretroviral therapy. N Engl J Med 1997;337:734–9. [2] Deeks SG, Lewin SR, Havlir DV. The end of AIDS: HIV infection as a chronic disease. Lancet 2013;382:1525–33. [3] Siliciano JD, Kajdas J, Finzi D, Quinn TC, Chadwick K, Margolick JB, et al. Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T cells. Nat Med 2003;9:727–8. [4] Svicher V, Ceccherini-Silberstein F, Antinori A, Aquaro S, Perno CF. Understanding HIV compartments and reservoirs. Curr HIV/AIDS Rep 2014;11:186–94. [5] Ho YC, Shan L, Hosmane NN, Wang J, Laskey SB, Rosenbloom DI, et al. Replication-competent noninduced proviruses in the latent reservoir increase barrier to HIV-1 cure. Cell 2013;155:540–51. [6] Hutter G, Nowak D, Mossner M, Ganepola S, Mussig A, Allers K, et al. Long-term control of HIV by CCR5 Delta32/ Delta32 stem-cell transplantation. N Engl J Med 2009;360: 692–8. [7] Allers K, Hutter G, Hofmann J, Loddenkemper C, Rieger K, Thiel E, et al. Evidence for the cure of HIV infection by CCR5Delta32/Delta32 stem cell transplantation. Blood 2011;117:2791–9. [8] de Goede AL, Vulto AG, Osterhaus AD, Gruters RA. Understanding HIV infection for the design of a therapeutic vaccine. Part I: epidemiology and pathogenesis of HIV infection. Ann Pharm Fr 2015;73:87–99.

HIV-resistant HSCT for a functional cure [9] Arts EJ, Hazuda DJ. HIV-1 antiretroviral drug therapy. Cold Spring Harb Perspect Med 2012;2,:a007161. [10] Abbas W, Tariq M, Iqbal M, Kumar A, Herbein G. Eradication of HIV-1 from the macrophage reservoir: an uncertain goal? Viruses 2015;7:1578–98. [11] Bruner KM, Hosmane NN, Siliciano RF. Towards an HIV-1 cure: measuring the latent reservoir. Trends Microbiol 2015;23:192–203. [12] Chun TW, Moir S, Fauci AS. HIV reservoirs as obstacles and opportunities for an HIV cure. Nat Immunol 2015; 16:584–9. [13] International AIDS Society Scientific Working Group on HIV Cure, Deeks SG, Autran B, Berkhout B, Benkirane M, Cairns S, et al. Towards an HIV cure: a global scientific strategy. Nat Rev Immunol 2012;12:607–14. [14] Rainwater-Lovett K, Luzuriaga K, Persaud D. Very early combination antiretroviral therapy in infants: prospects for cure. Curr Opin HIV AIDS 2015;10:4–11. [15] Liu R, Paxton WA, Choe S, Ceradini D, Martin SR, Horuk R, et al. Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell 1996;86:367–77. [16] Martinson JJ, Chapman NH, Rees DC, Liu YT, Clegg JB. Global distribution of the CCR5 gene 32-basepair deletion. Nat Genet 1997;16:100–3. [17] Henrich TJ, Hu Z, Li JZ, Sciaranghella G, Busch MP, Keating SM, et al. Long-term reduction in peripheral blood HIV type 1 reservoirs following reduced-intensity conditioning allogeneic stem cell transplantation. J Infect Dis 2013;207:1694–702. [18] Henrich TJ, Hanhauser E, Marty FM, Sirignano MN, Keating S, Lee TH, et al. Antiretroviral-free HIV-1 remission and viral rebound after allogeneic stem cell transplantation: report of 2 cases. Ann Intern Med 2014;161:319–27. [19] Persaud D, Gay H, Ziemniak C, Chen YH, Piatak M Jr, Chun TW, et al. Absence of detectable HIV-1 viremia after treatment cessation in an infant. N Engl J Med 2013;369:1828– 35. [20] Saez-Cirion A, Bacchus C, Hocqueloux L, Avettand-Fenoel V, Girault I, Lecuroux C, et al. Post-treatment HIV-1 controllers with a long-term virological remission after the interruption of early initiated antiretroviral therapy ANRS VISCONTI Study. PLoS Pathog 2013;9:e1003211. [21] Luzuriaga K, Gay H, Ziemniak C, Sanborn KB, Somasundaran M, Rainwater-Lovett K, et al. Viremic relapse after HIV-1 remission in a perinatally infected child. N Engl J Med 2015;372:786–8. [22] Archin NM, Margolis DM. Emerging strategies to deplete the HIV reservoir. Curr Opin Infect Dis 2014;27:29–35. [23] Kumar A, Darcis G, Van Lint C, Herbein G. Epigenetic control of HIV-1 post integration latency: implications for therapy. Clin Epigenetics 2015;7:103. [24] Liu C, Ma X, Liu B, Chen C, Zhang H. HIV-1 functional cure: will the dream come true? BMC Med 2015;13: 284. [25] Bullen CK, Laird GM, Durand CM, Siliciano JD, Siliciano RF. New ex vivo approaches distinguish effective and ineffective single agents for reversing HIV-1 latency in vivo. Nat Med 2014;20:425–9. [26] Barouch DH, Deeks SG. Immunologic strategies for HIV-1 remission and eradication. Science 2014;345:169–74. [27] Smith PL, Tanner H, Dalgleish A. Developments in HIV-1 immunotherapy and therapeutic vaccination. F1000Prime Rep 2014;6:43. [28] Kamphorst AO, Ahmed R. CD4 T-cell immunotherapy for chronic viral infections and cancer. Immunotherapy 2013;5:975–87.

1379

[29] Okoye AA, Picker LJ. CD4(+) T-cell depletion in HIV infection: mechanisms of immunological failure. Immunol Rev 2013;254:54–64. [30] Paiardini M, Muller-Trutwin M. HIV-associated chronic immune activation. Immunol Rev 2013;254:78–101. [31] Mehandru S, Poles MA, Tenner-Racz K, Horowitz A, Hurley A, Hogan C, et al. Primary HIV-1 infection is associated with preferential depletion of CD4+ T lymphocytes from effector sites in the gastrointestinal tract. J Exp Med 2004;200:761– 70. [32] Ussher JE, Klenerman P, Willberg CB. Mucosal-associated invariant T-cells: new players in anti-bacterial immunity. Front Immunol 2014;5:450. [33] Valverde-Villegas JM, Matte MC, de Medeiros RM, Chies JA. New insights about treg and Th17 cells in HIV infection and disease progression. J Immunol Res 2015;2015:647916. [34] Perez EE, Wang J, Miller JC, Jouvenot Y, Kim KA, Liu O, et al. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol 2008;26:808–16. [35] Holt N, Wang J, Kim K, Friedman G, Wang X, Taupin V, et al. Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo. Nat Biotechnol 2010;28:839–47. [36] Wilen CB, Wang J, Tilton JC, Miller JC, Kim KA, Rebar EJ, et al. Engineering HIV-resistant human CD4+ T cells with CXCR4-specific zinc-finger nucleases. PLoS Pathog 2011;7:e1002020. [37] Didigu CA, Wilen CB, Wang J, Duong J, Secreto AJ, Danet-Desnoyers GA, et al. Simultaneous zinc-finger nuclease editing of the HIV coreceptors ccr5 and cxcr4 protects CD4+ T cells from HIV-1 infection. Blood 2014;123:61–9. [38] van Lunzen J, Glaunsinger T, Stahmer I, von Baehr V, Baum C, Schilz A, et al. Transfer of autologous genemodified T cells in HIV-infected patients with advanced immunodeficiency and drug-resistant virus. Mol Ther 2007;15:1024–33. [39] Kimpel J, Braun SE, Qiu G, Wong FE, Conolle M, Schmitz JE, et al. Survival of the fittest: positive selection of CD4+ T cells expressing a membrane-bound fusion inhibitor following HIV-1 infection. PLoS ONE 2010;5: e12357. [40] Younan PM, Polacino P, Kowalski JP, Peterson CW, Maurice NJ, Williams NP, et al. Positive selection of mC46-expressing CD4+ T cells and maintenance of virus specific immunity in a primate AIDS model. Blood 2013;122:179–87. [41] Neagu MR, Ziegler P, Pertel T, Strambio-De-Castillia C, Grutter C, Martinetti G, et al. Potent inhibition of HIV-1 by TRIM5-cyclophilin fusion proteins engineered from human components. J Clin Invest 2009;119:3035–47. [42] Vets S, Kimpel J, Volk A, De Rijck J, Schrijvers R, Verbinnen B, et al. Lens epithelium-derived growth factor/p75 qualifies as a target for HIV gene therapy in the NSG mouse model. Mol Ther 2012;20:908–17. [43] Green M, Ishino M, Loewenstein PM. Mutational analysis of HIV-1 Tat minimal domain peptides: identification of trans-dominant mutants that suppress HIV-LTR-driven gene expression. Cell 1989;58:215–23. [44] Chatterjee S, Johnson PR, Wong KK Jr. Dual-target inhibition of HIV-1 in vitro by means of an adeno-associated virus antisense vector. Science 1992;258:1485–8. [45] Bevec D, Dobrovnik M, Hauber J, Bohnlein E. Inhibition of human immunodeficiency virus type 1 replication in human T cells by retroviral-mediated gene transfer of a dominant-negative Rev trans-activator. Proc Natl Acad Sci USA 1992;89:9870–4.

1380

J.Wang & M. C. Holmes

[46] Bahner I, Kearns K, Hao QL, Smogorzewska EM, Kohn DB. Transduction of human CD34+ hematopoietic progenitor cells by a retroviral vector expressing an RRE decoy inhibits human immunodeficiency virus type 1 replication in myelomonocytic cells produced in long-term culture. J Virol 1996;70:4352–60. [47] Arhel N, Kirchhoff F. Host proteins involved in HIV infection: new therapeutic targets. Biochim Biophys Acta 2010;1802:313–21. [48] Sarver N, Cantin EM, Chang PS, Zaia JA, Ladne PA, Stephens DA, et al. Ribozymes as potential anti-HIV-1 therapeutic agents. Science 1990;247:1222–5. [49] Tavassoli A, Lu Q, Gam J, Pan H, Benkovic SJ, Cohen SN. Inhibition of HIV budding by a genetically selected cyclic peptide targeting the Gag-TSG101 interaction. ACS Chem Biol 2008;3:757–64. [50] Lv M, Wang J, Zhu Y, Wang X, Zuo T, Liu D, et al. Overexpression of inactive tetherin delGPI mutant inhibits HIV-1 Vpu-mediated antagonism of endogenous tetherin. FEBS Lett 2013;587:37–43. [51] Mi Z, Wang X, He Y, Li X, Ding J, Liu H, et al. A novel peptide to disrupt the interaction of BST-2 and Vpu. Biopolymers 2014;102:280–7. [52] Burnett JC, Zaia JA, Rossi JJ. Creating genetic resistance to HIV. Curr Opin Immunol 2012;24:625–32. [53] Hoxie JA, June CH. Novel cell and gene therapies for HIV. Cold Spring Harb Perspect Med 2012;2. [54] Manjunath N, Yi G, Dang Y, Shankar P. Newer gene editing technologies toward HIV gene therapy. Viruses 2013;5:2748–66. [55] Li W, Yu M, Bai L, Bu D, Xu X. Downregulation of CCR5 expression on cells by recombinant adenovirus containing antisense CCR5, a possible measure to prevent HIV-1 from entering target cells. J Acquir Immune Defic Syndr 2006;43:516–22. [56] Tebas P, Stein D, Binder-Scholl G, Mukherjee R, Brady T, Rebello T, et al. Antiviral effects of autologous CD4 T cells genetically modified with a conditionally replicating lentiviral vector expressing long antisense to HIV. Blood 2013;121: 1524–33. [57] Wandtke T, Wozniak J, Kopinski P. Aptamers in diagnostics and treatment of viral infections. Viruses 2015;7:751–80. [58] Sullenger BA, Gallardo HF, Ungers GE, Gilboa E. Overexpression of TAR sequences renders cells resistant to human immunodeficiency virus replication. Cell 1990;63: 601–8. [59] Lee SW, Gallardo HF, Gilboa E, Smith C. Inhibition of human immunodeficiency virus type 1 in human T cells by a potent Rev response element decoy consisting of the 13-nucleotide minimal Rev-binding domain. J Virol 1994;68:8254–64. [60] DiGiusto DL, Krishnan A, Li L, Li H, Li S, Rao A, et al. RNA-based gene therapy for HIV with lentiviral vector-modified CD34(+) cells in patients undergoing transplantation for AIDS-related lymphoma. Sci Transl Med 2010;2:36ra43. [61] Ahlenstiel CL, Suzuki K, Marks K, Symonds GP, Kelleher AD. Controlling HIV-1: non-coding RNA gene therapy approaches to a functional cure. Front Immunol 2015;6:474. [62] Kumar P, Ban HS, Kim SS, Wu H, Pearson T, Greiner DL, et al. T cell-specific siRNA delivery suppresses HIV-1 infection in humanized mice. Cell 2008;134:577–86. [63] Ramakrishnan V. The ribosome emerges from a black box. Cell 2014;159:979–84. [64] Andang M, Hinkula J, Hotchkiss G, Larsson S, Britton S, Wong-Staal F, et al. Dose-response resistance to HIV-1/ MuLV pseudotype virus ex vivo in a hairpin ribozyme

[65]

[66]

[67]

[68]

[69]

[70]

[71]

[72]

[73]

[74]

[75]

[76] [77]

[78]

[79]

[80]

[81]

[82]

transgenic mouse model. Proc Natl Acad Sci USA 1999;96:12749–53. Yu M, Ojwang J, Yamada O, Hampel A, Rapapport J, Looney D, et al. A hairpin ribozyme inhibits expression of diverse strains of human immunodeficiency virus type 1. Proc Natl Acad Sci USA 1993;90:6340–4. Bai J, Gorantla S, Banda N, Cagnon L, Rossi J, Akkina R. Characterization of anti-CCR5 ribozyme-transduced CD34+ hematopoietic progenitor cells in vitro and in a SCID-hu mouse model in vivo. Mol Ther 2000;1:244–54. Kang EM, de Witte M, Malech H, Morgan RA, Phang S, Carter C, et al. Nonmyeloablative conditioning followed by transplantation of genetically modified HLA-matched peripheral blood progenitor cells for hematologic malignancies in patients with acquired immunodeficiency syndrome. Blood 2002;99:698–701. Steinberger P, Andris-Widhopf J, Buhler B, Torbett BE, Barbas CF 3rd. Functional deletion of the CCR5 receptor by intracellular immunization produces cells that are refractory to CCR5-dependent HIV-1 infection and cell fusion. Proc Natl Acad Sci USA 2000;97:805–10. Chen JD, Bai X, Yang AG, Cong Y, Chen SY. Inactivation of HIV-1 chemokine co-receptor CXCR-4 by a novel intrakine strategy. Nat Med 1997;3:1110–16. Yang AG, Bai X, Huang XF, Yao C, Chen S. Phenotypic knockout of HIV type 1 chemokine coreceptor CCR-5 by intrakines as potential therapeutic approach for HIV-1 infection. Proc Natl Acad Sci USA 1997;94:11567–72. Egelhofer M, Brandenburg G, Martinius H, Schult-Dietrich P, Melikyan G, Kunert R, et al. Inhibition of human immunodeficiency virus type 1 entry in cells expressing gp41-derived peptides. J Virol 2004;78:568–75. Leslie GJ, Wang J, Richardson M, Haggarty B, Hua KL, Duong J, et al. Potent and Broad Inhibition of HIV-1 by a Peptide from the gp41 Heptad Repeat-2 Domain Conjugated to the CXCR4 Amino Terminus. Submitted, 2016. Bhindi R, Fahmy RG, Lowe HC, Chesterman CN, Dass CR, Cairns MJ, et al. Brothers in arms: DNA enzymes, short interfering RNA, and the emerging wave of small-molecule nucleic acid-based gene-silencing strategies. Am J Pathol 2007;171:1079–88. Pandey VN, Upadhyay A, Chaubey B. Prospects for antisense peptide nucleic acid (PNA) therapies for HIV. Expert Opin Biol Ther 2009;9:975–89. Gaj T, Gersbach CA, Barbas CF 3rd. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 2013;31:397–405. Wyman C, Kanaar R. DNA double-strand break repair: all’s well that ends well. Annu Rev Genet 2006;40:363–83. San Filippo J, Sung P, Klein H. Mechanism of eukaryotic homologous recombination. Annu Rev Biochem 2008;77: 229–57. Wang J, Exline CM, DeClercq JJ, Llewellyn GN, Hayward SB, Li PW, et al. Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors. Nat Biotechnol 2015;33:1256–63. Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD. Genome editing with engineered zinc finger nucleases. Nat Rev Genet 2010;11:636–46. Wright DA, Li T, Yang B, Spalding MH. TALEN-mediated genome editing: prospects and perspectives. Biochem J 2014;462:15–24. Wright AV, Nunez JK, Doudna JA. Biology and applications of CRISPR systems: harnessing nature’s toolbox for genome engineering. Cell 2016;164:29–44. Li L, Krymskaya L, Wang J, Henley J, Rao A, Cao LF, et al. Genomic editing of the HIV-1 coreceptor CCR5 in adult

HIV-resistant HSCT for a functional cure

[83]

[84]

[85]

[86]

[87]

[88] [89]

[90]

[91]

[92]

[93]

[94]

[95]

[96]

[97]

[98]

[99]

hematopoietic stem and progenitor cells using zinc finger nucleases. Mol Ther 2013;21:1259–69. Cradick TJ, Fine EJ, Antico CJ, Bao G. CRISPR/Cas9 systems targeting beta-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res 2013;41: 9584–92. Kang H, Minder P, Park MA, Mesquitta WT, Torbett BE, Slukvin II. CCR5 disruption in induced pluripotent stem cells using CRISPR/Cas9 provides selective resistance of immune cells to CCR5-tropic HIV-1 virus. Mol Ther Nucleic Acids 2015;4:e268. Mock U, Machowicz R, Hauber I, Horn S, Abramowski P, Berdien B, et al. mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5. Nucleic Acids Res 2015;43:5560–71. Zhou Y, Kurihara T, Ryseck RP, Yang Y, Ryan C, Loy J, et al. Impaired macrophage function and enhanced T cell-dependent immune response in mice lacking CCR5, the mouse homologue of the major HIV-1 coreceptor. J Immunol 1998;160:4018–25. Yurchenko E, Tritt M, Hay V, Shevach EM, Belkaid Y, Piccirillo CA. CCR5-dependent homing of naturally occurring CD4+ regulatory T cells to sites of Leishmania major infection favors pathogen persistence. J Exp Med 2006;203:2451–60. Telenti A. Safety concerns about CCR5 as an antiviral target. Curr Opin HIV AIDS 2009;4:131–5. Kroetz DN, Deepe GS Jr. CCR5 dictates the equilibrium of proinflammatory IL-17+ and regulatory Foxp3+ T cells in fungal infection. J Immunol 2010;184:5224–31. Kroetz DN, Deepe GS Jr. An aberrant thymus in CCR5-/mice is coupled with an enhanced adaptive immune response in fungal infection. J Immunol 2011;186:5949–55. Huffnagle GB, McNeil LK, McDonald RA, Murphy JW, Toews GB, Maeda N, et al. Cutting edge: role of C-C chemokine receptor 5 in organ-specific and innate immunity to Cryptococcus neoformans. J Immunol 1999;163:4642–6. Chavez JH, Franca RF, Oliveira CJ, de Aquino MT, Farias KJ, Machado PR, et al. Influence of the CCR-5/MIP-1 alpha axis in the pathogenesis of Rocio virus encephalitis in a mouse model. Am J Trop Med Hyg 2013;89:1013–18. Galvani AP, Slatkin M. Evaluating plague and smallpox as historical selective pressures for the CCR5-Delta 32 HIV-resistance allele. Proc Natl Acad Sci USA 2003;100: 15276–9. Alonzo F 3rd, Kozhaya L, Rawlings SA, Reyes-Robles T, DuMont AL, Myszka DG, et al. CCR5 is a receptor for Staphylococcus aureus leukotoxin ED. Nature 2013;493:51–5. Elvin SJ, Williamson ED, Scott JC, Smith JN, Perez De Lema G, Chilla S, et al. Evolutionary genetics: ambiguous role of CCR5 in Y. pestis infection. Nature 2004;430:417. Mecsas J, Franklin G, Kuziel WA, Brubaker RR, Falkow S, Mosier DE. Evolutionary genetics: CCR5 mutation and plague protection. Nature 2004;427:606. Glass WG, McDermott DH, Lim JK, Lekhong S, Yu SF, Frank WA, et al. CCR5 deficiency increases risk of symptomatic West Nile virus infection. J Exp Med 2006;203:35–40. Kindberg E, Mickiene A, Ax C, Akerlind B, Vene S, Lindquist L, et al. A deletion in the chemokine receptor 5 (CCR5) gene is associated with tickborne encephalitis. J Infect Dis 2008;197:266–9. Yuan J, Wang J, Crain K, Fearns C, Kim KA, Hua KL, et al. Zinc-finger nuclease editing of human cxcr4 promotes HIV-1 CD4(+) T cell resistance and enrichment. Mol Ther 2012;20:849–59.

1381

[100] Zou YR, Kottmann AH, Kuroda M, Taniuchi I, Littman DR. Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature 1998;393:595–9. [101] Fruehauf S. Current clinical indications for plerixafor. Transfus Med Hemother 2013;40:246–50. [102] Eaves CJ. Hematopoietic stem cells: concepts, definitions, and the new reality. Blood 2015;125:2605–13. [103] Porada CD, Atala AJ, Almeida-Porada G. The hematopoietic system in the context of regenerative medicine. Methods 2015;99:44–61. [104] Lopez M, Beaujean F. Positive selection of autologous peripheral blood stem cells. Baillieres Best Pract Res Clin Haematol 1999;12:71–86. [105] Korbling M, Anderlini P. Peripheral blood stem cell versus bone marrow allotransplantation: does the source of hematopoietic stem cells matter? Blood 2001;98:2900–8. [106] Stan R, Zaia JA. Practical considerations in gene therapy for HIV cure. Curr HIV/AIDS Rep 2014;11:11–19. [107] Showel M, Fuchs EJ. Recent developments in HLAhaploidentical transplantations. Best Pract Res Clin Haematol 2015;28:141–6. [108] Tebas P, Stein D, Tang WW, Frank I, Wang SQ, Lee G, et al. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N Engl J Med 2014;370:901–10. [109] Kohn DB, Bauer G, Rice CR, Rothschild JC, Carbonaro DA, Valdez P, et al. A clinical trial of retroviral-mediated transfer of a rev-responsive element decoy gene into CD34(+) cells from the bone marrow of human immunodeficiency virus1-infected children. Blood 1999;94:368–71. [110] Amado RG, Mitsuyasu RT, Rosenblatt JD, Ngok FK, Bakker A, Cole S, et al. Anti-human immunodeficiency virus hematopoietic progenitor cell-delivered ribozyme in a phase I study: myeloid and lymphoid reconstitution in human immunodeficiency virus type-1-infected patients. Hum Gene Ther 2004;15:251–62. [111] Amado RG, Mitsuyasu RT, Symonds G, Rosenblatt JD, Zack J, Sun LQ, et al. A phase I trial of autologous CD34+ hematopoietic progenitor cells transduced with an anti-HIV ribozyme. Hum Gene Ther 1999;10:2255–70. [112] Michienzi A, Castanotto D, Lee N, Li S, Zaia JA, Rossi JJ. RNA-mediated inhibition of HIV in a gene therapy setting. Ann N Y Acad Sci 2003;1002:63–71. [113] Mitsuyasu RT, Merigan TC, Carr A, Zack JA, Winters MA, Workman C, et al. Phase 2 gene therapy trial of an anti-HIV ribozyme in autologous CD34+ cells. Nat Med 2009;15: 285–92. [114] Hayakawa J, Washington K, Uchida N, Phang O, Kang EM, Hsieh MM, et al. Long-term vector integration site analysis following retroviral mediated gene transfer to hematopoietic stem cells for the treatment of HIV infection. PLoS ONE 2009;4:e4211. [115] Podsakoff GM, Engel BC, Carbonaro DA, Choi C, Smogorzewska EM, Bauer G, et al. Selective survival of peripheral blood lymphocytes in children with HIV-1 following delivery of an anti-HIV gene to bone marrow CD34(+) cells. Mol Ther 2005;12:77–86. [116] Younan PM, Peterson CW, Polacino P, Kowalski JP, Obenza W, Miller HW, et al. Lentivirus-mediated Gene Transfer in Hematopoietic Stem Cells Is Impaired in SHIV-infected, ART-treated Nonhuman Primates. Mol Ther 2015;23:943– 51. [117] Beard BC, Trobridge GD, Ironside C, McCune JS, Adair JE, Kiem HP. Efficient and stable MGMT-mediated selection of long-term repopulating stem cells in nonhuman primates. J Clin Invest 2010;120:2345–54.