Adeno-associated Virus Transgene Research Articles

Sub-genomic flaviviral RNA elements increase the stability and abundance of recombinant AAV vector transcripts.

Viral RNA elements can hijack host cell machinery to control stability of transcripts and consequently, infection. Studies that help better understand such viral elements can provide insights into antiviral strategies and also potentially leverage these features for therapeutic applications. In this study, by incorporating structured flaviviral RNA elements into recombinant adeno-associated viral (AAV) vector genomes, we show improved AAV transcript stability and transgene expression can be achieved, with implications for gene transfer.

Essential role of pre-existing humoral immunity in TLR9-mediated type I IFN response to recombinant AAV vectors in human whole blood.

Recombinant adeno-associated virus (AAV) vectors have emerged as the preferred platform for gene therapy of rare human diseases. Despite the clinical promise, host immune responses to AAV vectors and transgene remain a major barrier to the development of successful AAV-based human gene therapies. Here, we assessed the human innate immune response to AAV9, the preferred serotype for AAV-mediated gene therapy of the CNS. We showed that AAV9 induced type I interferon (IFN) and IL-6 responses in human blood from healthy donors. This innate response was replicated with AAV6, required full viral particles, but was not observed in every donor. Depleting CpG motifs from the AAV transgene or inhibiting TLR9 signaling reduced type I IFN response to AAV9 in responding donors, highlighting the importance of TLR9-mediated DNA sensing for the innate response to AAV9. Remarkably, we further demonstrated that only seropositive donors with preexisting antibodies to AAV9 capsid mounted an innate immune response to AAV9 in human whole blood and that anti-AAV9 antibodies were necessary and sufficient to promote type I IFN release and plasmacytoid dendritic (pDC) cell activation in response to AAV9. Thus, our study reveals a previously unidentified requirement for AAV preexisting antibodies for TLR9-mediated type I IFN response to AAV9 in human blood.

TLR9-independent CD8+ T cell responses in hepatic AAV gene transfer through IL-1R1-MyD88 signaling

Upon viral infection of the liver, CD8+ Tcell responses may be triggered despite the immune suppressive properties that manifest in this organ. We sought to identify pathways that activate responses to a neoantigen expressed in hepatocytes, using adeno-associated viral (AAV) gene transfer. It was previously established that cooperation between plasmacytoid dendritic cells (pDCs), which sense AAV genomes by Toll-like receptor 9 (TLR9), and conventional DCs promotes cross-priming of capsid-specific CD8+ Tcells. Surprisingly, we find local initiation of a CD8+ Tcell response against antigen expressed in ∼20% of murine hepatocytes, independent of TLR9 or type I interferons and instead relying on IL-1 receptor 1-MyD88 signaling. Both IL-1α and IL-1β contribute to this response, which can be blunted by IL-1 blockade. Upon AAV administration, IL-1-producing pDCs infiltrate the liver and co-cluster with XCR1+ DCs, CD8+ Tcells, and Kupffer cells. Analogous events were observed following coagulation factor VIII gene transfer in hemophilia A mice. Therefore, pDCs have alternative means of promoting anti-viral Tcell responses and participate in intrahepatic immune cell networks similar to those that form in lymphoid organs. Combined TLR9 and IL-1 blockade may broadly prevent CD8+ T responses against AAV capsid and transgene product.

Chemical Epigenetic Regulation of Adeno-Associated Virus Delivered Transgenes.

Adeno-associated virus (AAV) is a powerful gene therapy vector that has been used in several FDA-approved therapies as well as in multiple clinical trials. This vector has high therapeutic versatility with the ability to deliver genetic payloads to a variety of human tissue types, yet there is currently a lack of transgene expression control once the virus is administered. There are also times when transgene expression is too low for the desired therapeutic outcome, necessitating high viral dose administration resulting in possible immunological complications. Herein, we validate a chemically controllable AAV transgene expression technology in vitro that utilizes bifunctional molecules known as chemical epigenetic modifiers (CEMs). These compounds employ endogenous epigenetic machinery to specifically enhance transgene expression of episomal DNA. A recombinant AAV (rAAV) was designed to both deliver the reporter transgene as well as deliver a synthetic zinc finger (ZFs) protein fused to FK506 binding protein (FKBP). These synthetic ZFs target a DNA-binding array sequence upstream of the promoter expressing the AAV transgene to specifically enhance AAV transgene expression in the presence of a CEM. The transcriptional activating compound CEM87 functions by recruiting the epigenetic transcription activator bromodomain-containing protein 4 (BRD4), increasing AAV transgene activity up to fivefold in a dose-dependent manner in HEK293T cells. The highest levels of transgene product activity are seen 24 h following CEM87 treatment. Additionally, the CEM87-mediated enhancement of different transgene products with either Luciferase or green fluorescent protein (GFP) was observed in multiple cell lines and enhancement of transgene expression was capsid serotype independent. The impact of CEM87 activity can be disrupted through drug removal or chemical recruitment site competition with FK506, thus demonstrating the reversibility of the impact of CEM87 on transgene expression. Collectively, this chemically controllable rAAV transgene technology provides temporal gene expression control that could increase the safety and efficiency of AAV-based research and therapies.

RNAi-mediated rheostat for dynamic control of AAV-delivered transgenes

Adeno-associated virus (AAV)-based gene therapy could be facilitated by the development of molecular switches to control the magnitude and timing of expression of therapeutic transgenes. RNA interference (RNAi)-based approaches hold unique potential as a clinically proven modality to pharmacologically regulate AAV gene dosage in a sequence-specific manner. We present a generalizable RNAi-based rheostat wherein hepatocyte-directed AAV transgene expression is silenced using the clinically validated modality of chemically modified small interfering RNA (siRNA) conjugates or vectorized co-expression of short hairpin RNA (shRNA). For transgene induction, we employ REVERSIR technology, a synthetic high-affinity oligonucleotide complementary to the siRNA or shRNA guide strand to reverse RNAi activity and rapidly recover transgene expression. For potential clinical development, we report potent and specific siRNA sequences that may allow selective regulation of transgenes while minimizing unintended off-target effects. Our results establish a conceptual framework for RNAi-based regulatory switches with potential for infrequent dosing in clinical settings to dynamically modulate expression of virally-delivered gene therapies.

Genome-Scale Analysis of Cellular Restriction Factors That Inhibit Transgene Expression from Adeno-Associated Virus Vectors.

Adeno-associated virus (AAV) vectors are one of the leading platforms for gene delivery for the treatment of human genetic diseases, but the antiviral cellular mechanisms that interfere with optimal transgene expression are incompletely understood. Here, we performed two genome-scale CRISPR screens to identify cellular factors that restrict transgene expression from recombinant AAV vectors. Our screens revealed several components linked to DNA damage response, chromatin remodeling, and transcriptional regulation. Inactivation of the Fanconi anemia gene FANCA; the human silencing hub (HUSH)-associated methyltransferase SETDB1; and the gyrase, Hsp90, histidine kinase, and MutL (GHKL)-type ATPase MORC3 led to increased transgene expression. Moreover, SETDB1 and MORC3 knockout improved transgene levels of several AAV serotypes as well as other viral vectors, such as lentivirus and adenovirus. Finally, we demonstrated that the inhibition of FANCA, SETDB1, or MORC3 also enhanced transgene expression in human primary cells, suggesting that they could be physiologically relevant pathways that restrict AAV transgene levels in therapeutic settings. IMPORTANCE Recombinant AAV (rAAV) vectors have been successfully developed for the treatment of genetic diseases. The therapeutic strategy often involves the replacement of a defective gene by the expression of a functional copy from the rAAV vector genome. However, cells possess antiviral mechanisms that recognize and silence foreign DNA elements thereby limiting transgene expression and its therapeutic effect. Here, we utilize a functional genomics approach to uncover a comprehensive set of cellular restriction factors that inhibit rAAV-based transgene expression. Genetic inactivation of selected restriction factors increased rAAV transgene expression. Hence, modulation of identified restriction factors has the potential to enhance AAV gene replacement therapies.

Transcriptome analysis of Sf9 insect cells during production of recombinant Adeno-associated virus.

The insect cell-baculovirus expression vector system (IC-BEVS) has emerged as an alternative time- and cost-efficient production platform for recombinant Adeno-associated virus (AAV) for gene therapy. However, a better understanding of the underlying biological mechanisms of IC-BEVS is fundamental to further optimize this expression system toward increased product titer and quality. Here, gene expression of Sf9 insect cells producing recombinant AAV through a dual baculovirus expression system, with low multiplicity of infection (MOI), was profiled by RNA-seq. An 8-fold increase in reads mapping to either baculovirus or AAV transgene sequences was observed between 24 and 48h post-infection (hpi), confirming a take-over of the host cell transcriptome by the baculovirus. A total of 336 and 4784 genes were identified as differentially expressed at 24 hpi (vs non-infected cells) and at 48 hpi (vs. infected cells at 24 hpi), respectively, including dronc, birc5/iap5, and prp1. Functional annotation found biological processes such as cell cycle, cell growth, protein folding, and cellular amino acid metabolic processes enriched along infection. This work uncovers transcriptional changes in Sf9 in response to baculovirus infection, which provide new insights into cell and/or metabolic engineering targets that can be leveraged for rational bioprocess engineering of IC-BEVS for AAV production.

Key Role of Kupffer Cells in IL-1 Dependent Activation of CD8+ T Cell Responses to AAV Transgene Product in Liver

Introduction: Adeno-associated virus (AAV) based viral vectors have emerged as preferred vectors for in vivo gene therapy and are being evaluated in multiple phase I/II and phase III clinical studies. However, immune responses to both the AAV capsid and its encoded transgene still remain a major hurdle. Previously, we demonstrated in a murine model that cellular responses to AAV encoded transgene in the liver are dependent on IL-1α/β mediated activation of IL-1R1-MyD88 signaling. Using flow cytometry and tissue staining we provided evidence that plasmacytoid dendritic cells (pDCs) and Kupffer cells are central players in initiation of IL-1 signaling. Further, we showed that cross-presenting XCR1+ dendritic cells (DCs) are critical for antigen presentation to transgene-specific CD8+ T cells (Blood 136 Supplement 1: 2-3, 2020). In the present study we tested the impact of Kupffer cells depletion on transgene-specific CD8+ T cell response. Since Kupffer cells have been implicated in TLR4-MyD88 mediated secretion of IL-1α in a murine model of acetaminophen induced liver injury (Cell Mol Immunol 15(11): 973, 2018). Therefore, we also explored if innate sensor TLR4 is required for IL-1 mediated CD8+ T cell response to AAV encoded transgene in liver microenvironment. Further, we tested if intrinsic IL-1R1-MyD88 signaling in DCs is essential for these cellular responses. Methods: To study the impact of Kupffer cell depletion on transgene-specific CD8+ T cells we intraperitoneally injected wild-type (WT) C57BL6 mice with 200μg of depleting α-mouse F4/80 antibody (BioXcell) one day prior and 2X/week following vector administered for up to 4 weeks. Groups of C57BL6 WT (control), C57BL6 WT (treated with α-mouse F4/80 antibody) and TLR4-/- mice on the C57BL6 background were intravenously (IV) injected with a predetermined immunogenic dose (1x109vg) of hepatotropic AAV8 vector encoding ovalbumin (OVA) as the transgene (Mol Ther 25(4): 880, 2017). To study the role of MyD88 intrinsic signaling in DCs, we generated mice that lacked adaptor protein MyD88 in DCs (DC-MyD88-/-) using cre-lox system (Blood 129(24): 3184, 2017). Mice were again injected with 1x109vg of AAV8-OVA via IV route. Four weeks after vector administration PBMCs from each group were analyzed for OVA-specific CD8+ T cells using MHC class I tetramer. Results: As expected, ~62% of WT mice developed OVA-specific CD8+ T cell response. Interestingly, only 12% of mice (1 out of 8) treated with α-mouse F4/80 antibody showed CD8+ T cells specific to OVA. Whereas similar to WT mice, 50% of TLR4-/- mice had OVA-specific CD8+ T cell. We conclude that Kupffer cells, which show increased surface expression of IL-1R1 within days after AAV vector administration, are essential for the IL-1R-dependent CD8+ T cells response to the hepatic transgene product but do not function through a mechanism that requires TLR4. This mechanism therefore shares some aspects of acetaminophen-induced liver injury (critical role of KCs and IL-1) but is distinct with regard to TLR involvement. IL-1R1 signaling requires the cytosolic MyD88 adapter molecule. We know from prior studies that MHC I antigen presentation of liver-derived AAV encoded transgene product in lymphoid organs is primarily mediated by cross-presenting XCR1+ DCs. Thus, we tested the requirement of intrinsic MyD88 signaling in DCs. Our results suggest that intrinsic MyD88 signaling in DCs is not required in lymphoid organs, as the CD8+ T cell response in DC-MyD88-/- mice was comparable to that in WT control mice (these mice lack MyD88 in conventional DCs in lymphoid organs but retain MyD88 in hepatic DCs and in pDCs). Conclusions: In summary, we established that Kupffer cells play a central role in elicitation of transgene product-specific CD8+ T cell responses in hepatic AAV gene transfer (through a TLR4-independent mechanisms). While direct sensing of IL-1 by DCs in the hepatic environment may very well play a role, it is not required in lymphoid organs. Following initial priming of CD8+ T cell in the liver, co-stimulation by CD4+ T helper cells may replace IL-1 signaling during the subsequent systemic response.

Gene therapy - are we ready now?

IntroductionHaemophilia therapy has evolved from rudimentary transfusion‐based approaches to an unprecedented level of innovation with glimmers of functional cure brought by gene therapy. After decades of misfires, gene therapy has normalized factor (F)VIII and factor (F)IX levels in some individuals in the long term. Several clinical programmes testing adeno‐associated viral (AAV) vector gene therapy are approaching completion with imminent regulatory approvals.DiscussionPhase 3 studies along with multiyear follow‐up in earlier phase investigations raised questions about efficacy as well as short‐ and long‐term safety, prompting a reappraisal of AAV vector gene therapy. Liver toxicities, albeit mostly low‐grade, occur in the first year in at least some individuals in all haemophilia A and B trials and are poorly understood. Extreme variability and unpredictability of outcome, as well as a slow decline in factor expression (seemingly unique to FVIII gene therapy), are vexing because immune responses to AAV vectors preclude repeat dosing, which could increase suboptimal or restore declining expression, while overexpression may result in phenotoxicity. The long‐term safety will need lifelong monitoring because AAV vectors, contrary to conventional wisdom, integrate into chromosomes at the rate that calls for vigilance.ConclusionsAAV transduction and transgene expression engage the host immune system, cellular DNA processing, transcription and translation machineries in ways that have been only cursorily studied in the clinic. Delineating those mechanisms will be key to finding mitigants and solutions to the remaining problems, and including individuals who cannot avail of gene therapy at this time.

Tropism of AAV Vectors in Photoreceptor-Like Cells of Human iPSC-Derived Retinal Organoids.

PurposeTo expand the use of human retinal organoids from induced pluripotent stem cells (iPSCs) as an in vitro model of the retina for assessing gene therapy treatments, it is essential to establish efficient transduction. To date, targeted transduction of the photoreceptor-like cells of retinal organoids with adeno-associated virus (AAV) vectors has had varied degrees of success, which we have looked to improve in this study.MethodsRetinal organoids were differentiated from iPSCs of healthy donors and transduced with reporter AAV containing a CAG.GFP, CAG.RFP, GRK1.GFP, or EFS.GFP transgene. Capsid variants assessed were AAV5, AAV2 7m8, AAV2 quad mutant, AAV2 Y444F, and AAV8 Y733F. At 27 days post-transduction, retinal organoids were assessed for reporter expression and viability.ResultsThe short intron-less elongation factor 1 alpha (EFS) promoter provided minimal reporter expression, whereas vectors containing the CAG promoter enabled transduction in 1% to 37% of cells depending on the AAV serotype; the AAV2 quad mutant (average 19.4%) and AAV2 7m8 (16.4%) outperformed AAV5 (12%) and AAV8 Y733F (2.1%). Reporter expression from rhodopsin kinase (GRK1) promoter transgenes occurred in ∼5% of cells regardless of the serotype. Positive co-localization with recoverin-expressing cells was achieved from all GRK1 vectors and the CAG AAV2 quad mutant variant. Treatment with the AAV vectors did not influence retinal organoid viability.ConclusionsReliable transduction of the photoreceptor-like cells of retinal organoids can be readily achieved. When using a CAG-driven transgene, transduction of a broad range of cell types is observed, and GRK1 transgenes provide a more restricted expression profile locating to the outer layer of photoreceptor-like cells of retinal organoids.Translational RelevanceThis study expands the AAV capsid and transgene options for preclinical testing of gene therapy in iPSC-derived human retinal organoids.

Systemic and local immune responses to intraocular AAV vector administration in non-human primates

Positive clinical outcomes in adeno-associated virus (AAV)-mediated retinal gene therapy have often been attributed to the low immunogenicity of AAVs and immune privilege of the eye. However, several recent studies have shown potential for inflammatory responses. The current understanding of the factors contributing to inflammation, such as the pre-existence of serum antibodies against AAVs and their contribution to increases in antibody levels post-injection, is incomplete. The parameters that regulate the generation of new antibodies in response to the AAV capsid or transgene after intraocular injections are also insufficiently described. This study is a retrospective analysis of the pre-existing serum antibodies in correlation with changes in antibody levels after intraocular injections of AAV in non-human primates (NHPs) of the species Macaca fascicularis. In NHP serums, we analyzed the binding antibody (BAB) levels and a subset of these called neutralizing antibodies (NABs) that impede AAV transduction. We observed significantly higher pre-existing serum BABs against AAV8 compared with other serotypes and a dose-dependent increase in BABs and NABs in the serums collected post-injection, irrespective of the serotype or the mode of injection. Lastly, we were able to demonstrate a correlation between the serum BAB levels with clinical grading of inflammation and levels of transgene expression.

Dexamethasone Transiently Enhances Transgene Expression in the Liver When Administered at Late-Phase Post Long-Term Adeno-Associated Virus Transduction.

Glucocorticoids have anti-inflammatory and immunosuppressive functions and have commonly been used for preventing liver toxicity after the systemic application of a high dose of adeno-associated virus (AAV) vector for gene therapy. Clinical studies have reported that glucocorticoids have rescued factor IX (FIX) expression in patients with hemophilia B who showed a reduced FIX expression at 6 to 10 weeks post-AAV vector administration. In this study, we explored whether glucocorticoids could affect transgene expression in AAV targeted livers in animal models. When dexamethasone was applied before AAV9/FIX vector administration in the wild-type C57BL/6 mice, FIX expression was much higher than that of the control mice at any time point. More importantly, FIX expression transiently increased after dexamethasone was administered at week 6 or later post-AAV injection regardless of the various dexamethasone treatments applied. The transient enhancement in transgene expression was observed once there were one to several consecutive dexamethasone treatments completed. A similar result was also achieved in other wild-type BALB/c and hemophilia B mice that were treated with AAV9/FIX and dexamethasone. This mechanism study demonstrated that the administration of dexamethasone did not change either AAV genome copy number or transgene expression at the transcription level but transiently decreased interferon beta (IFN-β) and tumor necrosis factor alpha (TNF-α) expression in the livers of mice at a later time after AAV injection. Next, we studied the effect of dexamethasone on late transgene expression in hemophilia B dogs. Dexamethasone was administered 1 year after AAV9/FIX injection. Inconsistent with the results in mice, no significant change of FIX expression was observed in hemophilia B dogs. In summary, the results from this study indicate that dexamethasone may have various effects on transgene expression in AAV-transduced livers in different species, which provides valuable information about the rational application of dexamethasone in future clinical studies.

ImmTOR nanoparticles enhance AAV transgene expression after initial and repeat dosing in a mouse model of methylmalonic acidemia

A major barrier to adeno-associated virus (AAV) gene therapy is the inability to re-dose patients due to formation of vector-induced neutralizing antibodies (Nabs). Tolerogenic nanoparticles encapsulating rapamycin (ImmTOR) provide long-term and specific suppression of adaptive immune responses, allowing for vector re-dosing. Moreover, co-administration of hepatotropic AAV vectors and ImmTOR leads to an increase of transgene expression even after the first dose. ImmTOR and AAV Anc80 encoding the methylmalonyl-coenzyme A (CoA) mutase (MMUT) combination was tested in a mouse model of methylmalonic acidemia, a disease caused by mutations in the MMUT gene. Repeated co-administration of Anc80 and ImmTOR was well tolerated and led to nearly complete inhibition of immunoglobulin (Ig)G antibodies to the Anc80 capsid. A more profound decrease of plasma levels of the key toxic metabolite, plasma methylmalonic acid (pMMA), and disease biomarker, fibroblast growth factor 21 (FGF21), was observed after treatment with the ImmTOR and Anc80-MMUT combination. In addition, there were higher numbers of viral genomes per cell (vg/cell) and increased transgene expression when ImmTOR was co-administered with Anc80-MMUT. These effects were dose-dependent, with the higher doses of ImmTOR providing higher vg/cell and mRNA levels, and an improved biomarker response. Combining of ImmTOR and AAV can not only block the IgG response against capsid, but it also appears to potentiate transduction and enhance therapeutic transgene expression in the mouse model.

Research Highlights

Research Highlights

An InVitro Whole-Organ Liver Engineering for Testing of Genetic Therapies.

SummaryExplosion of gene therapy approaches for treating rare monogenic and common liver disorders created an urgent need for disease models able to replicate human liver cellular environment. Available models lack 3D liver structure or are unable to survive in long-term culture. We aimed to generate and test a 3D culture system that allows long-term maintenance of human liver cell characteristics.The in vitro whole-organ “Bioreactor grown Artificial Liver Model” (BALM) employs a custom-designed bioreactor for long-term 3D culture of human induced pluripotent stem cells-derived hepatocyte-like cells (hiHEPs) in a mouse decellularized liver scaffold. Adeno-associated viral (AAV) and lentiviral (LV) vectors were introduced by intravascular injection.Substantial AAV and LV transgene expression in the BALM-grown hiHEPs was detected. Measurement of secreted proteins in the media allowed non-invasive monitoring of the system.We demonstrated that humanized whole-organ BALM is a valuable tool to generate pre-clinical data for investigational medicinal products.

Experimental Variables that Affect Human Hepatocyte AAV Transduction in Liver Chimeric Mice

Adeno-associated virus (AAV) vector serotypes vary in their ability to transduce hepatocytes from different species. Chimeric mouse models harboring human hepatocytes have shown translational promise for liver-directed gene therapies. However, many variables that influence human hepatocyte transduction and transgene expression in such models remain poorly defined. Here, we aimed to test whether three experimental conditions influence AAV transgene expression in immunodeficient, fumaryl-acetoactetate-hydrolase-deficient (Fah−/−) chimeric mice repopulated with primary human hepatocytes. We examined the effects of the murine liver injury cycle, human donor variability, and vector doses on hepatocyte transduction with various AAV serotypes expressing a green fluorescent protein (GFP). We determined that the timing of AAV vector challenge in the liver injury cycle resulted in up to 7-fold differences in the percentage of GFP expressing human hepatocytes. The GFP+ hepatocyte frequency varied 7-fold between human donors without, however, changing the relative transduction efficiency between serotypes for an individual donor. There was also a clear relationship between AAV vector doses and human hepatocyte transduction and transgene expression. We conclude that several experimental variables substantially affect human hepatocyte transduction in the Fah−/− chimera model, attention to which may improve reproducibility between findings from different laboratories.

Recombinant Adeno-Associated Viral Vectors Expressing Human Coagulation FIX-E456H Variant in Hemophilia B Mice.

Gene therapy using recombinant adeno-associated virus (AAV) has induced sustained long-term coagulation human factor IX (hFIX) levels in hemophilia B (HB) patients. However, asymptomatic transient liver toxicity was observed at high vector doses, highlighting the need to improve the potency of these vectors. We report the generation of an AAV transgene cassette containing the hyperfunctional hFIX-E456H variant showing improved binding to platelets, with a comparison to wild-type hFIX (hFIX-WT) and hFIX-R384L variant (Padua) transgenes, containing F9 truncated-intron 1 (I1). In vitro specific activity was increased by 3.2- and 4.2-fold with hFIX-E456H and hFIX-R384L variants compared with hFIX-WT, using chromogenic assay, and by 7-and 8.6-fold with hFIX-E456H and hFIX-R384L variants compared with hFIX-WT, using one-stage assay. The transgenes were packaged into single-stranded AAV2/8 vectors that were tail vein injected at 5 × 109, 2 × 1010, and 5 × 1010 vg per mouse in HB mice. Plasma FIX activity level, assessed by chromogenic assay, was up to fourfold higher for hFIX-E456H compared with hFIX-WT and was not different compared with hFIX-R384L, among the three dose cohorts. Overall, the in vivo specific activity was increased by threefold for hFIX-E456H and 4.9-fold for hFIX-R384L compared with hFIX-WT. At the lower dose of 5 × 109 vg, the blood loss was significantly lower for hFIX-E456H compared with hFIX-WT, but did not differ compared with hFIX-R384L. The results found for the hFIX-E456H variant indicate that it might be a suitable alternative for gene therapy of HB.

Advances of adeno-associated virus applied in gene therapy to hemophilia from bench work to the clinical use.

Hemophilia A and B are diseases caused by a single gene deficiency and are thus suitable for gene therapy. In recent clinical research, adeno-associated virus (AAV) was employed by several teams in the treatment of hemophilia A and B, and the outcomes were encouraging. In this review, we summarized the most recent research on the mechanism and application of AAV in the treatment of hemophilia, trying to analyze the advantages of AAV gene therapy and the main challenges in its clinical use. We also summarized the clinical trials involving hemophilia, especially those employing AAV gene therapy to treat hemophilia A and B, some of which have already been completed and some that are still ongoing. From the reports of the completed clinical trials, we tried to determine the correlations among AAV dose, AAV serotype, immune response, and gene expression time. Finally, taking into account the most recent studies investigating AAV capsid modification, transgene optimization, and AAV chaperones, we summarized the direction of basic research and clinical applications of AAV in the future.

An AAV Dual Vector Strategy Ameliorates the Stargardt Phenotype in Adult Abca4-/- Mice.

The recent approval in the United States of the first adeno-associated viral (AAV) vector for the treatment of an inherited retinal degeneration validates this approach for the treatment of many other diseases. A major limiting factor continues to be the size restriction of the AAV transgene at under 5 kb. Stargardt disease is the most prevalent form of recessively inherited blindness and is caused by mutations in ABCA4, the gene that codes for ATP-binding cassette transporter protein family member 4, which has a coding sequence length of 6.8 kb. Dual vector approaches increase the capacity of AAV gene therapy, but at the cost of substantially reduced levels of target protein, which may be insufficient to achieve a therapeutic effect. Here we show that the efficacy of recombination of dual vectors is dependent on the length of DNA overlap between two transgenes. With optimized recombination, full-length ABCA4 protein is expressed in the photoreceptor outer segments of Abca4-/- mice at levels sufficient to reduce bisretinoid formation and correct the autofluorescent phenotype. These observations support a dual vector approach in future clinical trials using AAV gene therapy to treat Stargardt disease.

AAV8 Gene Therapy for Crigler-Najjar Syndrome in Macaques Elicited Transgene T Cell Responses That Are Resident to the Liver

Systemic delivery of adeno-associated viral (AAV) vectors has been evaluated for the treatment of several liver diseases, including homozygous familial hypercholesterolemia, ornithine transcarbamylase deficiency, and hemophilia. Here, we evaluated this approach for the treatment of Crigler-Najjar syndrome. We administered wild-type rhesus macaques with 1.0 × 1013 or 2.5 × 1013 genome copies/kg of an AAV serotype 8 vector expressing a codon-optimized version of human uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1) from a liver-specific promoter. We extensively studied vector biodistribution, transgene expression, and immune responses following vector administration. All rhesus macaques survived until their scheduled necropsy at day 56 and showed no clinical abnormalities during the course of the study. Macaques administered with either vector dose developed a T cell response to the AAV capsid and/or transgene. We mapped the immunodominant epitope in the human UGT1A1 sequence, and we found no correlation between peripheral and tissue-resident lymphocyte responses. Upon further investigation, we characterized CD107a+, granzyme B+, CD4+, and CD8+ transgene-specific cellular responses that were restricted to tissue-resident T cells. This study highlights the importance of studying immune responses at the vector transduction site and the limited usefulness of blood as a surrogate to evaluate tissue-restricted T cell responses.