Adeno-associated Virus Gene Therapy Research Articles

Transcriptome of HPβCD-treated Niemann-Pick disease type C1 cells highlights GPNMB as a biomarker for therapeutics

The rare, fatal neurodegenerative disorder Niemann-Pick disease type C1 (NPC1) arises from lysosomal accumulation of unesterified cholesterol and glycosphingolipids. These subcellular pathologies lead to phenotypes of hepatosplenomegaly, neurological degeneration and premature death. The timing and severity of NPC1 clinical presentation is extremely heterogeneous. This study analyzed RNA-Seq data from 42 NPC1 patient-derived, primary fibroblast cell lines to determine transcriptional changes induced by treatment with 2-hydroxypropyl-β-cyclodextrin (HPβCD), a compound currently under investigation in clinical trials. A total of 485 HPβCD-responsive genes were identified. Pathway enrichment analysis of these genes showed significant involvement in cholesterol and lipid biosynthesis. Furthermore, immunohistochemistry of the cerebellum as well as measurements of plasma from Npc1m1N null mice treated with HPβCD and adeno-associated virus gene therapy suggests that one of the identified genes, GPNMB, may serve as a useful biomarker of treatment response in NPC1 disease. Overall, this large NPC1 patient-derived dataset provides a comprehensive foundation for understanding the genomic response to HPβCD treatment.

Development of a stable lyophilized adeno-associated virus gene therapy formulation

Adeno-associated viruses (AAV) are among the most actively investigated vectors for gene therapy. Supply of early clinical studies with frozen drug product (DP) can accelerate timelines and minimize degradation risks. In the long-term, logistical challenges of frozen DP may limit patient access. In this work, we developed a lyophilized (freeze-dried) formulation of AAV. The mass concentration of AAV is typically low, and AAV also requires a minimum ionic strength to inhibit aggregation. These factors result in a low collapse temperature, which is limiting to lyophilization. Mannitol crystallization was found to cause extensive degradation and potency loss of AAV during the freezing step. With further development, we determined that AAV could be lyophilized in a sucrose and citrate formulation with a more desirable high glass transition temperature of the dried cake. An optimal residual moisture range (1–3%) was found to be critical to maintaining AAV8 stability. Glycerol was found to protect AAV8 from over-drying by preventing capsid damage and genome DNA release. A lyophilized formulation was identified that maintained potency for 24 months at 2–8 °C, indicating the feasibility of a dried formulation for AAV gene therapy.

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.

Production of Adeno-Associated Virus Vectors in Cell Stacks for Preclinical Studies in Large Animal Models.

Adeno-associated virus (AAV) vectors are among the most clinically advanced gene therapy vectors, with three AAV gene therapies approved for humans. Clinical advancement of novel applications for AAV involves transitioning from small animal models, such as mice, to larger animal models, including dogs, sheep, and nonhuman primates. One of the limitations of administering AAV to larger animals is the requirement for large quantities of high-titer virus. While suspension cell culture is a scalable method for AAV vector production, few research labs have the equipment (e.g., bioreactors) or know how to produce AAV in this manner. Moreover, AAV titers are often significantly lower when produced in suspension HEK 293 cells as compared to adherent HEK293 cells. Described here is a method for producing large quantities of high-titer AAV using cell stacks. A detailed protocol for titering AAV as well as methods for validating vector purity are also described. Finally, representative results of AAV-mediated transgene expression in a sheep model are presented. This optimized protocol for large-scale production of AAV vectors in adherent cells will enable molecular biology laboratories to advance the testing of their novel AAV therapies in larger animal models.

101HEMB01 Is a Phase 1/2 Open-Label, Single Ascending Dose-Finding Trial of DTX101 (AAVrh10FIX) in Patients with Moderate/Severe Hemophilia B That Demonstrated Meaningful but Transient Expression of Human Factor IX (hFIX)

▪Background: Gene transfer is expected to be an effective therapy for hemophilia B because the disease is caused by mutations in a single gene. DTX101 is a non-replicating recombinant adeno-associated virus (AAV) serotype rh10 (AAVrh10) vector that contains a codon-optimized hFIX gene with expression driven by both a liver-specific enhancer element and a liver-specific promoter element. AAVrh10 is a clade E virus with high degree of homology to AAV8.Aims: To determine the safety and efficacy of single, ascending doses of DTX101 in hemophilia B patients.Methods: 101HEMB01 is a phase 1/2 open-label, single-arm, multicenter, dose-finding study. Eligible subjects (Inclusion criteria: Male ≥18 years, baseline FIX ≤2%; Exclusion criteria: anti-AAVrh10 neutralizing antibody titer >1:5) received a single IV infusion of DTX101. Safety assessments included incidence of adverse events (AEs). Efficacy assessments included change from baseline in FIX activity, annualized bleeding rate and annualized use of FIX replacement therapy.Results: 6 subjects were dosed. Subjects in Cohort 1 (n=3) and Cohort 2 (n=3) received a dose of DTX101 of 1.6×1012 Genome Copies (GC)/kg and 5.0×1012 GC/kg, respectively. All subjects have been followed ≥32 weeks. Peak FIX levels in Cohort 1 were achieved between 8 and 14 weeks and ranged from 5% - 11%. Peak FIX levels in Cohort 2 ranged from 12% - 20% and were reached between 3 and 8 weeks. An asymptomatic rise in alanine aminotransferase (ALT) level was observed at approximately the time of peak FIX level and was followed by a gradual reduction in FIX level to near baseline in all but one subject, despite initiation of a steroid treatment protocol. Peak ALT values were considered either Grade 1 or 2 AEs in 4 subjects (≤3.0 or ≤5.0 x upper limit of normal, respectively). Subject 6 had a peak ALT 914 U/L and was considered a Grade 4 AE. Steroid protocol was initiated with normalization of ALT levels. Subjects 4 and 5 had subsequent mild rises in ALT following completion of the steroid protocol with additional loss in FIX activity. No subject developed FIX inhibitors. Bleeding events and use of FIX replacement therapy data are currently being analyzed.Conclusion: Study 101HEMB01 clearly demonstrated evidence of FIX expression following a single injection of DTX101 (peak FIX 5% - 20%); however, FIX levels were not sustained. The loss in FIX levels may be related to AAVrh10 capsid-induced immune response; Dimension Therapeutics has ongoing studies to better understand this response (ASH 2017 abstract submission: R. Calcedo, Immune responses in 101HEMB01, a phase 1/2 open-label, single ascending dose-finding trial of DTX101 (AAVrh10FIX) in patients with severe hemophilia B). The 6 subjects dosed in study 101HEMB01 will continue to be closely followed for a total of 5 years in the extension study (101HEMB02). Insights from these data support continued development of other AAV gene therapy programs. DisclosuresPipe:Bayer Healthcare; Biogen Idec; CSL Behring; Dimension Therapeutics; Novo Nordisk; Roche; Shire; uniQure: Consultancy; Shire; Siemens Healthcare Diagnostics Inc.: Research Funding. Poma:Dimension Therapeutics: Employment, Equity Ownership. Crombez:Dimension Therapeutics: Employment, Equity Ownership.

GENE THERAPY FOR HAEMOPHILIA

The blood disorder, Hemophilia, has its roots embedded deep into the history of genetic disorders. The European royal family is one of the most prominent families to be affected by this disease thus, dubbing it 'the royal disease'. The types of Hemophilia are divided into two based on the type of coagulation factor mutation found in the patient. For treating haemophilia, gene therapy is done by using different vectors such as lentiviral and retroviral vectors but due to the production of limited expression different adeno associated virus (AAV) strains are used. Some engineerly modified vectors are currently used to get the best possible results. The clinical trials prove the efficacy of these vectors so through their obtained statistical consideration, patient experience and population study once can design vaccines and drugs for haemophilia patients but also due to pre-existing Nabs and pre-existing HCV or HBV infection, the general application of AAV gene therapy is currently limited. The possibility of gene editing for the repair of the mutation is on the horizon.

Adeno-associated viral (AAV) vector-mediated therapeutics for diabetic cardiomyopathy - current and future perspectives.

Diabetes increases the prevalence of heart failure by 6–8-fold, independent of other comorbidities such as hypertension and coronary artery disease, a phenomenon termed diabetic cardiomyopathy. Several key signalling pathways have been identified that drive the pathological changes associated with diabetes-induced heart failure. This has led to the development of multiple pharmacological agents that are currently available for clinical use. While fairly effective at delaying disease progression, these treatments do not reverse the cardiac damage associated with diabetes. One potential alternative avenue for targeting diabetes-induced heart failure is the use of adeno-associated viral vector (AAV) gene therapy, which has shown great versatility in a multitude of disease settings. AAV gene therapy has the potential to target specific cells or tissues, has a low host immune response and has the possibility to represent a lifelong cure, not possible with current conventional pharmacotherapies. In this review, we will assess the therapeutic potential of AAV gene therapy as a treatment for diabetic cardiomyopathy.

Adeno-associated virus gene therapy to the rescue for Charcot-Marie-Tooth disease type 4J.

The genetic peripheral neuropathy known as Charcot-Marie-Tooth disease type 4J (CMT4J) is caused by recessive mutations in the FIG4 gene. The transformational success of adeno-associated virus (AAV) gene therapy for spinal muscular atrophy has generated substantial interest in using this approach to create similar treatments for CMT. In this issue of the JCI, Presa et al. provide a preclinical demonstration of efficacy using AAV-directed gene therapy for CMT4J. The study showed a dramatic improvement in both survival and neuropathy symptoms in a severe mouse model of CMT4J after administration of AAV gene therapy at several time points. The authors' approach advances the technique for delivering treatments to individuals with CMT, for which FDA-approved therapies have not yet come to the clinic.

Central nervous system-targeted adeno-associated virus gene therapy in methylmalonic acidemia

Methylmalonic acidemia (MMA) is a severe metabolic disorder most commonly caused by a mutation in the methylmalonyl-CoA mutase (MMUT) gene. Patients with MMA experience multisystemic disease manifestations and remain at risk for neurological disease progression, even after liver transplantation. Therefore, delivery of MMUT to the central nervous system (CNS) may provide patients with neuroprotection and, perhaps, therapeutic benefits. To specifically target the brain, we developed a neurotropic PHP.eB vector that used a CaMKII neuro-specific promoter to restrict the expression of the MMUT transgene in the neuraxis and delivered the adeno-associated virus (AAV) to mice with MMA. The PHP.eB vector transduced cells in multiple brain regions, including the striatum, and enabled high levels of expression of MMUT in the basal ganglia. Following the CNS-specific correction of MMUT expression, disease-related metabolites methylmalonic acid and 2-methylcitrate were significantly (p < 0.02) decreased in serum of treated MMA mice. Our results show that targeting MMUT expression to the CNS using a neurotropic capsid can decrease the circulating metabolite load in MMA and further highlight the benefit of extrahepatic correction for disorders of organic acid metabolism.

Effect of CpG Depletion of Vector Genome on CD8+ T Cell Responses in AAV Gene Therapy.

Adeno associated viral (AAV) vectors have emerged as a preferred platform for in vivo gene replacement therapy and represent one of the most promising strategies to treat monogenetic disorders such as hemophilia. However, immune responses to gene transfer have hampered human gene therapy in clinical trials. Over the past decade, it has become clear that innate immune recognition provides signals for the induction of antigen-specific responses against vector or transgene product. In particular, TLR9 recognition of the vector’s DNA genome in plasmacytoid dendritic cells (pDCs) has been identified as a key factor. Data from clinical trials and pre-clinical studies implement CpG motifs in the vector genome as drivers of immune responses, especially of CD8+ T cell activation. Here, we demonstrate that cross-priming of AAV capsid-specific CD8+ T cells depends on XCR1+ dendritic cells (which are likely the main cross-presenting cell that cooperates with pDCs to activate CD8+ T cells) and can be minimized by the elimination of CpG motifs in the vector genome. Further, a CpG-depleted vector expressing human coagulation factor IX showed markedly reduced (albeit not entirely eliminated) CD8+ T cell infiltration upon intramuscular gene transfer in hemophilia B mice when compared to conventional CpG+ vector (comprised of native sequences), resulting in better preservation of transduced muscle fibers. Therefore, this deimmunization strategy is helpful in reducing the potential for CD8+ T cell responses to capsid or transgene product. However, CpG depletion had minimal effects on antibody responses against capsid or transgene product, which appear to be largely independent of CpG motifs.

Monitoring cell-mediated immune responses in AAV gene therapy clinical trials using a validated IFN-γ ELISpot method

Adeno-associated virus (AAV)-based gene therapies have recently shown promise as a novel treatment for hereditary diseases. Due to the viral origin of the vector capsid, however, cellular immune response may be elicited that could eliminate transduced target cells. To monitor cellular immune responses in clinical trials, we optimized and bioanalytically validated a sensitive, robust, and reliable interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) assay. For method performance validation, human peripheral blood mononuclear cells (PBMCs) were stimulated with peptides derived from AAV5 capsid proteins and the encoded transgene product, human blood clotting factor VIII (FVIII), in addition to positive controls, such as peptides from the 65-kDa phosphoprotein of cytomegalovirus. We statistically assessed the limit of detection and confirmatory cutpoint, evaluated precision and linearity, and confirmed specificity using HIV peptides. Robustness parameter ranges and sample stability periods were established. The validated IFN-γ ELISpot assay was then implemented in an AAV5-FVIII gene therapy clinical trial. Cellular immune responses against the AAV5 capsid were observed in most participants as soon as 2 weeks following dose administration; only limited responses against the transgene product were detected. These data underscore the value of using validated methods for monitoring cellular immunity in AAV gene therapy trials.

Humoral immune responses to AAV gene therapy in the ocular compartment.

Viral vectors can be utilised to deliver therapeutic genes to diseased cells. Adeno-associated virus (AAV) is a commonly used viral vector that is favoured for its ability to infect a wide range of tissues whilst displaying limited toxicity and immunogenicity. Most humans harbour anti-AAV neutralising antibodies (NAbs) due to subclinical infections by wild-type virus during infancy and these pre-existing NAbs can limit the efficiency of gene transfer depending on the target cell type, route of administration and choice of serotype. Vector administration can also result in de novo NAb synthesis that could limit the opportunity for repeated gene transfer to diseased sites. A number of strategies have been described in preclinical models that could circumvent NAb responses in humans, however, the successful translation of these innovations into the clinical arena has been limited. Here, we provide a comprehensive review of the humoral immune response to AAV gene therapy in the ocular compartment. We cover basic AAV biology and clinical application, the role of pre-existing and induced NAbs, and possible approaches to overcoming antibody responses. We conclude with a framework for a comprehensive strategy for circumventing humoral immune responses to AAV in the future.

Anti-AAV Antibodies in AAV Gene Therapy: Current Challenges and Possible Solutions.

Adeno-associated virus (AAV) vector-based gene therapy is currently the only in vivo gene therapy approved in the US and Europe. The recent tragic death of three children in a clinical trial to treat X-Linked Myotubular Myopathy by delivering myotubularin with an AAV8 vector notwithstanding, AAV remains a highly promising therapeutic gene delivery platform. But the successful use of AAV vectors to treat an increasing number of diseases also makes establishing protocols to determine therapeutically relevant titers of pre-existing anti-AAV antibodies and approaches to deplete those antibodies more urgent than ever. In this mini review, I will briefly discuss (i) our knowledge regarding the prevalence of anti-AAV antibodies, (ii) the challenges to measure those antibodies by methods that are most predictive of their influence on therapeutic efficacy of AAV gene transfer, and (iii) approaches to overcome the formidable hurdle that anti-AAV antibodies pose to the successful clinical use of AAV gene therapy.

Adeno-associated Virus (AAV) Capsid Chimeras with Enhanced Infectivity Reveal a Core Element in the AAV Genome Critical for both Cell Transduction and Capsid Assembly.

Adeno-associated viruses (AAV) have attracted significant attention in the field of gene and cell therapy due to highly effective delivery of therapeutic genes into human cells. The ability to generate recombinant AAV vectors compromised of unique or substituted protein sequences has led to the development of capsid variants with improved therapeutic properties. Seeking novel AAV vectors capable of enhanced transduction for therapeutic applications, we have developed a series of unique capsid variants termed AAV X-Vivo (AAV-XV) derived from chimeras of AAV12 VP1/2 sequences and the VP3 sequence of AAV6. These AAV variants showed enhanced infection of human primary T cells, hematopoietic stem cells, and neuronal cell lines over wildtype parental viruses, and superiority over AAV6 for genomic integration of DNA sequences by AAV alone or in combination with CRISPR gene editing. AAV-XV variants demonstrate transduction efficiency equivalent to AAV6 at multiplicities of infection 2 logs lower, enabling T cell engineering at low AAV doses. The protein coding sequence of these novel AAV chimeras revealed disruptions within the assembly-activating protein (AAP) which likely accounted for observed lower virus yield. A series of genome alterations, reverting the AAP sequence back to wildtype AAV6, had a negative impact on the enhanced transduction seen with AAV-VX, indicating overlapping functions within this sequence for both viral assembly and effective T cell transduction. Our findings show these AAV-XV variants are highly efficient at cell transduction at low dose and demonstrates the importance of the AAP coding region in both viral particle assembly and cell infection.IMPORTANCE A major hurdle to the therapeutic potential of AAV in gene therapy lies in achieving clinically meaningful AAV doses, and secondarily, ability to manufacture commercially viable titers of AAV to support this. By virtue of neutralizing antibodies against AAV that impede patient repeat-dosing, the dose of AAV for in vivo gene delivery has been high, which has resulted in unfortunate recent safety concerns and deaths in patients given higher-dose AAV gene therapy. We have generated new AAV variants possessing unique combinations of capsid proteins for gene and cell therapy applications termed AAV-XV, which have high levels of cell transduction and gene delivery at lower MOI. Furthermore, we demonstrate a novel finding, and an important consideration for recombinant AAV design, that a region of the AAV genome encoding the capsid viral protein and AAP is critical for both virus yield and the enhancement of infection/transduction.

Intracellular trafficking of adeno-associated virus (AAV) vectors: challenges and future directions.

In the last two decades, recombinant adeno-associated virus has emerged as the most popular gene therapy vector. Recently AAV gene therapy has been approved by the FDA for the treatment of two rare genetic disorders, namely the early childhood blindness disease Leber congenital amaurosis and spinal muscular atrophy (SMA). As is the case for the treatment of SMA, if the AAV vector must be administered systemically, very high vector doses are often required for therapeutic efficacy. But higher vector doses inevitably increase the risk of adverse events. The tragic death of three children in a clinical trial to treat X-linked myotubular myopathy with an AAV vector has thrown this limitation into sharp relief. Regardless of the precise cause(s) that led to the death of the two children, it is critical that we develop better AAV vectors to achieve therapeutic levels of expression with lower vector doses. To transduce successfully a target cell, AAV has to overcome both systemic as well as cellular roadblocks. In this review, we discuss some of the most prominent cellular roadblocks that AAV must get past to deliver successfully its therapeutic payload. We also highlight recent advancements in our knowledge of AAV biology that can potentially be harnessed to improve AAV vector performance and thereby make AAV gene therapy safer.

Gene Therapy of Dominant CRX-Leber Congenital Amaurosis using Patient Stem Cell-Derived Retinal Organoids.

SummaryMutations in the photoreceptor transcription factor gene cone-rod homeobox (CRX) lead to distinct retinopathy phenotypes, including early-onset vision impairment in dominant Leber congenital amaurosis (LCA). Using induced pluripotent stem cells (iPSCs) from a patient with CRX-I138fs48 mutation, we established an in vitro model of CRX-LCA in retinal organoids that showed defective photoreceptor maturation by histology and gene profiling, with diminished expression of visual opsins. Adeno-associated virus (AAV)-mediated CRX gene augmentation therapy partially restored photoreceptor phenotype and expression of phototransduction-related genes as determined by single-cell RNA-sequencing. Retinal organoids derived from iPSCs of a second dominant CRX-LCA patient carrying K88N mutation revealed the loss of opsin expression as a common phenotype, which was alleviated by AAV-mediated augmentation of CRX. Our studies provide a proof-of-concept for developing gene therapy of dominant CRX-LCA and other CRX retinopathies.

AAV Joins the Rank of Genotoxic Vectors

AAV Joins the Rank of Genotoxic Vectors

Demonstrating Comparability of AAV Gene Therapy Products During Clinical Development: Managing the Link Between the Product and the Process

While the ideal path during product development would be to avoid process changes, especially during later phases of clinical development, manufacturing changes are common in gene therapy products for many reasons. Development of a comparability strategy should be incorporated during product development to minimize risks to future approvals. This comparability strategy may include non-clinical and/or clinical bridging studies to support the claim for comparability, as part of a stepwise approach, but an increased understanding of the physicochemical attributes of the Adeno-Associated Virus (AAV)-based product and understanding the structure-function relationships for the product may pre-empt the need for such bridging studies. To meet this objective, the results of the comparability assessment should demonstrate that there has been no change in the structure-function relationship of the product and that there is no ambiguity in the results. In this article, the authors suggest key points for consideration when planning and executing the comparability assessment.

Overcoming innate immune barriers that impede AAV gene therapy vectors.

The field of gene therapy has made considerable progress over the past several years. Adeno-associated virus (AAV) vectors have emerged as promising and attractive tools for in vivo gene therapy. Despite the recent clinical successes achieved with recombinant AAVs (rAAVs) for therapeutics, host immune responses against the vector and transgene product have been observed in numerous preclinical and clinical studies. These outcomes have hampered the advancement of AAV gene therapies, preventing them from becoming fully viable and safe medicines. The human immune system is multidimensional and complex. Both the innate and adaptive arms of the immune system seem to play a concerted role in the response against rAAVs. While most efforts have been focused on the role of adaptive immunity and developing ways to overcome it, the innate immune system has also been found to have a critical function. Innate immunity not only mediates the initial response to the vector, but also primes the adaptive immune system to launch a more deleterious attack against the foreign vector. This Review highlights what is known about innate immune responses against rAAVs and discusses potential strategies to circumvent these pathways.

Progress and Challenges in Clinical AAV Gene Therapy for Neurological and Neuromuscular Disorders

The field of gene therapy is growing at a rapid pace. The discovery and modification of adeno-associated viruses (AAV) as gene therapy vectors has allowed for the development of novel treatments for neurological and neuromuscular disorders. AAV based gene therapy techniques can be utilized to add, replace, or modify genes and their expression in patients, thus providing a potential beneficial therapeutic effect. In many cases, gene therapy has the promising power to change the natural course of debilitating and fatal diseases and give patients a new outcome on life. This mini review will give a brief overview of the AAV gene therapy field, highlighting FDA approved gene therapies and selected current in-clinic AAV-mediated therapeutics for neurological and neuromuscular diseases for which interim data are available. These therapies and the ongoing clinical trials are currently laying the groundwork for future gene therapy strategies and clinical trial design. Ultimately, they will inform the field on promises and limitations that still need to be overcome.