Adeno-associated Virus Gene Transfer Research Articles

The Impact of Pre-existing Immunity on the Non-clinical Pharmacodynamics of AAV5-Based Gene Therapy

Adeno-associated virus (AAV)-based vectors are widely used for gene therapy, but the effect of pre-existing antibodies resulting from exposure to wild-type AAV is unclear. In addition, other poorly defined plasma factors could inhibit AAV vector transduction where antibodies are not detected. To better define the relationship between various forms of pre-existing AAV immunity and gene transfer, we studied valoctocogene roxaparvovec (BMN 270) in cynomolgus monkeys with varying pre-dose levels of neutralizing anti-AAV antibodies and non-antibody transduction inhibitors. BMN 270 is an AAV5-based vector for treating hemophilia A that encodes human B domain-deleted factor VIII (FVIII-SQ). After infusion of BMN 270 (6.0 × 1013 vg/kg) into animals with pre-existing anti-AAV5 antibodies, there was a mean decrease in maximal FVIII-SQ plasma concentration (Cmax) and AUC of 74.8% and 66.9%, respectively, compared with non-immune control animals, and vector genomes in the liver were reduced. In contrast, animals with only non-antibody transduction inhibitors showed FVIII-SQ plasma concentrations and liver vector copies comparable with those of controls. These results demonstrate that animals without AAV5 antibodies are likely responders to AAV5 gene therapy, regardless of other inhibiting plasma factors. The biological threshold for tolerable AAV5 antibody levels varied between individual animals and should be evaluated further in clinical studies.

Gene therapy for C1 esterase inhibitor deficiency in a Murine Model of Hereditary angioedema.

Hereditary angioedema (HAE) is a life-threatening, autosomal dominant disorder characterized by unpredictable, episodic swelling of the face, upper airway, oropharynx, extremities, genitalia, and gastrointestinal tract. Almost all cases of HAE are caused by mutations in the SERPING1 gene resulting in a deficiency in functional plasma C1 esterase inhibitor (C1EI), a serine protease inhibitor that normally inhibits proteases in the contact, complement, and fibrinolytic systems. Current treatment of HAE includes long-term prophylaxis with attenuated androgens or human plasma-derived C1EI and management of acute attacks with human plasma-derived or recombinant C1EI, bradykinin, and kallikrein inhibitors, each of which requires repeated administration. As an approach to effectively treat HAE with a single treatment, we hypothesized that a one-time intravenous administration of an adeno-associated virus (AAV) gene transfer vector expressing the genetic sequence of the normal human C1 esterase inhibitor (AAVrh.10hC1EI) would provide sustained circulating C1EI levels sufficient to prevent angioedema episodes. To study the efficacy of AAVrh.10hC1EI, we used CRISPR/Cas9 technology to create a heterozygote C1EI-deficient mouse model (S63±) that shares characteristics associated with HAE in humans including decreased plasma C1EI and C4 levels. Phenotypically, these mice have increased vascular permeability of skin and internal organs. Systemic administration of AAVrh.10hC1EI to the S63± mice resulted in sustained human C1EI activity levels above the predicted therapeutic levels and correction of the vascular leak in skin and internal organs. A single treatment with AAVrh.10hC1EI has the potential to provide long-term protection from angioedema attacks in affected individuals.

Gene-based FVIIa prophylaxis modulates the spontaneous bleeding phenotype of hemophilia A rats

AbstractA sizable proportion of hemophilia inhibitor patients fails immune tolerance induction and requires bypass agents for long-term bleed management. Recombinant human-activated coagulation Factor VII (rhFVIIa) is an on-demand bypass hemostatic agent for bleeds in hemophilia inhibitor patients. Prophylactic use of rhFVIIa may enable sustained hemostatic management of inhibitor patients, but the critical relationship of rhFVIIa circulating levels and clinical outcome in that setting remains unclear. To address this in vivo, we used the rat hemophilia A (HA) model that exhibits spontaneous bleeds and allows longitudinal studies with sufficient statistical power. We simulated activated Factor VII (FVIIa) prophylaxis by adeno-associated virus (AAV) gene transfer of a rat FVIIa transgene. Compared with naive HA animals, rat FVIIa continuous expression affected the overall observed bleeds, which were resolved with on-demand administration of recombinant rat FVIIa. Specifically, although 91% of naive animals exhibited bleeds, this was reduced to 83% and 33% in animals expressing less than 708 ng/mL (<14 nM) and at least 708 ng/mL (≥14 nM) rat FVIIa, respectively. No bleeds occurred in animals expressing higher than 1250 ng/mL (>25 nM). Rat FVIIa expression of at least 708 ng/mL was also sufficient to normalize the blood loss after a tail vein injury. Continuous, AAV-mediated rat FVIIa transgene expression had no apparent adverse effects in the hemostatic system of HA rats. This work establishes for the first time a dose dependency and threshold of circulating FVIIa antigen levels for reduction or complete elimination of bleeds in a setting of FVIIa-based HA prophylaxis.

Knockdown of CLC-3 in the hippocampal CA1 impairs contextual fear memory

Previous studies support a critical role of hippocampus in contextual fear memory. Structural and functional alterations of hippocampus occur frequently in posttraumatic stress disorders (PTSD). Recent reports reveal that knockout of CLC-3, a member of the CLC family of anion channels and transporters, leads to neuronal degeneration and loss of hippocampus. However, the role of CLC-3 in contextual fear memory remains unknown. Using adenovirus and adeno-associated virus gene transfer to knockdown CLC-3 in hippocampal CA1, we investigate the role of CLC-3 in contextual fear memory. CLC-3 expression is increased in hippocampal CA1 after formation of long-term contextual fear memory. Knockdown of CLC-3 by adenovirus infusion in hippocampal CA1 significantly attenuates the contextual fear memory, reduces spine density, induces defects of excitatory synaptic ultrastructure showed by the decreased PSD length, PSD thickness and active zone length, and impairs L-LTP induction and maintenance. Knockdown of CLC-3 also induces the synaptic NMDAR subunit composition to an increased GluN2A/GluN2B ratio pattern and reduces the activity of CaMKII-α. Furthermore, selectively knockdown of CLC-3 in excitatory neurons by adeno-associated virus driven from CaMKII-α promoter is sufficient to impair long-term contextual fear memory. These findings highlight that CLC-3 in hippocampal CA1 is necessary for contextual fear memory.

Fetal gene therapy for neurodegenerative disease of infants.

For inherited genetic diseases, fetal gene therapy offers the potential of prophylaxis against early, irreversible and lethal pathological change. To explore this, we studied neuronopathic Gaucher disease (nGD), caused by mutations in GBA. In adult patients, the milder form presents with hepatomegaly, splenomegaly and occasional lung and bone disease; this is managed, symptomatically, by enzyme replacement therapy. The acute childhood lethal form of nGD is untreatable since enzyme cannot cross the blood-brain barrier. Patients with nGD exhibit signs consistent with hindbrain neurodegeneration, including neck hyperextension, strabismus and, often, fatal apnea1. We selected a mouse model of nGD carrying a loxP-flanked neomycin disruption of Gba plus Cre recombinase regulated by the keratinocyte-specific K14 promoter. Exclusive skin expression of Gba prevents fatal neonatal dehydration. Instead, mice develop fatal neurodegeneration within 15 days2. Using this model, fetal intracranial injection of adeno-associated virus (AAV) vector reconstituted neuronal glucocerebrosidase expression. Mice lived for up to at least 18 weeks, were fertile and fully mobile. Neurodegeneration was abolished and neuroinflammation ameliorated. Neonatal intervention also rescued mice but less effectively. As the next step to clinical translation, we also demonstrated the feasibility of ultrasound-guided global AAV gene transfer to fetal macaque brains.

Gene Therapy induced Tregs: A treatment for relapsing-remitting MS in mice

Abstract Autoimmune disease is caused by a breakdown of tolerance against endogenous protein(s). New therapies are being developed that utilize the natural abilities of regulatory T (Treg) cells to suppress autoimmune effector cells in an antigen-specific manner. Recently, we demonstrated a clinically relevant in vivo gene immunotherapy approach for restoring Ag-specific tolerance using hepatic adeno-associated virus (AAV) gene transfer. This approach was shown to be remarkably successful at preventing and reversing a monophasic, MOG-induced EAE disease. Additionally, we have shown the dynamic ability of this treatment to prevent and/or reverse disease induced with various MOG epitopes, in genetically diverse mouse strains. However, it is unclear if this approach would be effective in the more complicated, PLP induced relapsing-remitting disease model. Thus, we re-engineered our therapeutic vector to contain the full-coding sequence of PLP. This is critically important as the established RR-EAE (SJL) model is characterized by epitope spreading; a phenomenon that may more closely resemble multiple sclerosis. SJL mice received a single peripheral injection of AAV. PLP vector, or control, 2 weeks prior to PLP 139–151 induced EAE. By day 14, control mice developed significant neurological deficits followed by a relapsing-remitting disease course. In contrast, the treated mice failed to exhibit any neurological symptoms of disease throughout the duration of the experiment, ~75 days. The long-term prevention of PLP induced disease, provides the first evidence of an effective Treg based therapy for combating epitope drift in RR-EAE, and potentially a new paradigm for treating the autoimmune disease multiple sclerosis.

Non-invasive detection of adeno-associated viral gene transfer using a genetically encoded CEST-MRI reporter gene in the murine heart

Research into gene therapy for heart failure has gained renewed interest as a result of improved safety and availability of adeno-associated viral vectors (AAV). While magnetic resonance imaging (MRI) is standard for functional assessment of gene therapy outcomes, quantitation of gene transfer/expression relies upon tissue biopsy, fluorescence or nuclear imaging. Imaging of gene expression through the use of genetically encoded chemical exchange saturation transfer (CEST)-MRI reporter genes could be combined with clinical cardiac MRI methods to comprehensively probe therapeutic gene expression and subsequent outcomes. The CEST-MRI reporter gene Lysine Rich Protein (LRP) was cloned into an AAV9 vector and either administered systemically via tail vein injection or directly injected into the left ventricular free wall of mice. Longitudinal in vivo CEST-MRI performed at days 15 and 45 after direct injection or at 1, 60 and 90 days after systemic injection revealed robust CEST contrast in myocardium that was later confirmed to express LRP by immunostaining. Ventricular structure and function were not impacted by expression of LRP in either study arm. The ability to quantify and link therapeutic gene expression to functional outcomes can provide rich data for further development of gene therapy for heart failure.

Preclinical models to assess the immunogenicity of AAV vectors

Although gene transfer using adeno-associated virus (AAV) vectors has made tremendous progress in recent years, challenges remain due to vector-specific adaptive immune responses. Specifically, AAV-neutralizing antibodies reduce AAV-transduction rates, while CD8+ T cells directed to AAV capsid antigens cause rejection of AAV-transduced cells. This has been addressed clinically by excluding humans with pre-existing AAV-neutralizing antibodies from gene transfer trials or by using immunosuppression or reduced doses of vectors expressing improved transgene products to blunt or circumvent destructive T cell responses. Although these approaches have met with success for treatment of some diseases, most notably hemophilia B, they may not be suitable for others. Pre-clinical models are thus needed to test alternative options to sidestep pre-existing AAV-neutralizing antibodies, to prevent their induction following gene transfer and to block the detrimental effects of CD8+ T cells directed to AAV capsid antigens. This chapter describes some of the available, although not yet perfect, models that can assess immune responses to AAV gene transfer.

Covalent coupling of high-affinity ligands to the surface of viral vector particles by protein trans-splicing mediates cell type-specific gene transfer.

We have established a novel approach for the covalent coupling of large polypeptides to the surface of fully assembled adeno-associated viral gene transfer vector (AAV) particles via split-intein mediated protein-trans-splicing (PTS). This way, we achieved selective gene transfer to distinct cell types. Single-chain variable fragments (scFvs) or designed ankyrin repeat proteins (DARPins), exhibiting high-affinity binding to cell surface receptors selectively expressed on the surface of target cells, were coupled to AAV particles harboring mutations in the capsid proteins which ablate natural receptor usage. Both, the AAV capsid protein VP2 and multiple separately produced targeting ligands recognizing Her2/neu, EpCAM, CD133 or CD30 were genetically fused with complementary split-intein domains. Optimized coupling conditions led to an effective conjugation of each targeting ligand to the universal AAV capsid and translated into specific gene transfer into target receptor-positive cell types invitro and invivo. Interestingly, PTS-based AAVs exhibited significantly less gene transfer into target receptor-negative cells than AAVs displaying the same targeting ligand but coupled genetically. Another important consequence of the PTS technology is the possibility to now display scFvs or other antibody-derived domain formats harboring disulfide-bonds in a functionally active form on the surface of AAV particles. Hence, the custom combination of a universal AAV vector particle and targeting ligands of various formats allows for an unprecedented flexibility in the generation of gene transfer vectors targeted to distinct cell types.

An Immune-Competent Murine Model to Study Elimination of AAV-Transduced Hepatocytes by Capsid-Specific CD8+ T Cells

Multiple independent adeno-associated virus (AAV) gene therapy clinical trials for hemophilia B, utilizing different AAV serotypes, have reported a vector dose-dependent loss of circulating factor IX (FIX) protein associated with capsid-specific CD8+ T cell (Cap-CD8) elimination of transduced hepatocytes. Hemophilia B patients who develop transient transaminitis and loss of FIX protein may be stabilized with the immune-suppressive (IS) drug prednisolone, but do not all recover lost FIX expression, whereas some patients fail to respond to IS. We developed the first animal model demonstrating Cap-CD8 infiltration and elimination of AAV-transduced hepatocytes of immune-deficient mice. Here, we extend this model to an immune-competent host where Cap-CD8 transfer to AAV2-F9-treated mice significantly reduced circulating and hepatocyte FIX expression. Further, we studied two high-expressing liver tropic AAV2 variants, AAV2-LiA and AAV2-LiC, obtained from a rationally designed capsid library. Unlike AAV2, Cap-CD8 did not initially reduce circulating FIX levels for either variant. However, FIX levels were significantly reduced in AAV2-LiC-F9-treated, but not AAV2-LiA-F9-treated, mice at the study endpoint. Going forward, the immune-competent model may provide an opportunity to induce immunological memory directed against a surrogate AAV capsid antigen and study recall responses following AAV gene transfer.

Liver-Specific Allergen Gene Transfer by Adeno-Associated Virus Suppresses Allergic Airway Inflammation in Mice.

Allergic airway inflammation driven by T helper 2 (Th2)-type immunity is characterized by airway hyperresponsiveness, eosinophilic infiltration, and elevated IgE production. Various novel strategies for managing asthma have been explored, such as DNA vaccines, T-cell peptides, and allergen-specific immunotherapy. A principal goal of most immunotherapeutic approaches is active and long-term allergen-specific tolerance. Liver-specific gene transfer using adeno-associated virus (AAV) has been shown to favorably induce tolerogenic responses to therapeutic products in various experimental models. AAV8 has strong liver tropism and induces immune tolerance in mice. The present study aimed to determine whether hepatocyte-specific allergen expression by pseudotyped AAV2/8 alleviates asthmatic symptoms in ovalbumin (OVA)-sensitized mice. Mice were intravenously injected with AAV2/8 vector carrying membrane-bound OVA transgene under transcriptional control of a hepatocyte-specific alpha 1 antitrypsin promoter (AAV2/8-OVA) and then sensitized with OVA. AAV2/8-OVA specifically transduced the OVA transgene in the liver. Airway hyperresponsiveness, eosinophilia, mucus hypersecretion, and Th2 cytokines were significantly suppressed in both the lungs and secondary lymphoid organs of asthmatic mice infected with AAV2/8-OVA. Significant reduction of OVA-specific antibodies was detected in the bronchoalveolar lavage fluid from AAV2/8-OVA-treated mice. Moreover, AAV2/8-OVA treatment prominently promoted the expression of Foxp3, IL-10, and TGF-β in the liver. Enhanced Foxp3 expression was also detected in the lungs of asthmatic mice after AAV2/8-OVA treatment. Taken together, these results suggest that the induction of immune tolerance by hepatic AAV gene transfer may be beneficial for modulating allergic asthma.

Insulin Therapy Improves Adeno-Associated Virus Transduction of Liver and Skeletal Muscle in Mice and Cultured Cells.

Adeno-associated virus (AAV) gene transfer is a promising treatment for genetic abnormalities. Optimal AAV vectors are showing success in clinical trials. Gene transfer to skeletal muscle and liver is being explored as a potential therapy for some conditions, that is, α1-antitrypsin (AAT) disorder and hemophilia B. Exploring approaches that enhance transduction of liver and skeletal muscle, using these vectors, is beneficial for gene therapy. Regulating hormones as an approach to improve AAV transduction is largely unexplored. In this study we tested whether insulin therapy improves liver and skeletal muscle gene transfer. In vitro studies demonstrated that the temporary coadministration (2, 8, and 24 hr) of insulin significantly improves AAV2-CMV-LacZ transduction of cultured liver cells and differentiated myofibers, but not of lung cells. In addition, there was a dose response related to this improved transduction. Interestingly, when insulin was not coadministered with the virus but given 24 hr afterward, there was no increase in the transgene product. Insulin receptor gene (INSR) expression levels were increased 5- to 13-fold in cultured liver cells and differentiated myofibers when compared with lung cells. Similar INSR gene expression profiles occurred in mouse tissues. Insulin therapy was performed in mice, using a subcutaneously implanted insulin pellet or a high-carbohydrate diet. Insulin treatment began just before intramuscular delivery of AAV1-CMV-schFIX or liver-directed delivery of AAV8-CMV-schFIX and continued for 28 days. Both insulin augmentation therapies improved skeletal muscle- and liver-directed gene transduction in mice as seen by a 3.0- to 4.5-fold increase in human factor IX (hFIX) levels. The improvement was observed even after the insulin therapy ended. Monitoring insulin showed that insulin levels increased during the brief period of rAAV delivery and during the entire insulin augmentation period (28 days). This study demonstrates that AAV transduction of liver or skeletal muscle can be improved by insulin therapy.

Anti-hIgE gene therapy of peanut-induced anaphylaxis in a humanized murine model of peanut allergy

Peanuts are the most common food to provoke fatal or near-fatal anaphylactic reactions. Treatment with an anti-hIgE mAb is efficacious but requires frequent parenteral administration. Based on the knowledge that peanut allergy is mediated by peanut-specific IgE, we hypothesized that a single administration of an adeno-associated virus (AAV) gene transfer vector encoding for anti-hIgE would protect against repeated peanut exposure in the host with peanut allergy. We developed a novel humanized murine model of peanut allergy that recapitulates the human anaphylactic response to peanuts in NOD-scid IL2Rgammanull mice transferred with blood mononuclear cells from donors with peanut allergy and then sensitized with peanut extract. As therapy, we constructed an adeno-associated rh.10 serotype vector coding for a full-length, high-affinity, anti-hIgE antibody derived from the Fab fragment of the anti-hIgE mAb omalizumab (AAVrh.10anti-hIgE). In the reconstituted mice peanut-specific IgE was induced by peanut sensitization and hypersensitivity, and reactions were provoked by feeding peanuts to mice with symptoms similar to those of human subjects with peanut allergy. A single administration of AAVrh.10anti-hIgE vector expressed persistent levels of anti-hIgE. The anti-hIgE vector, administered either before sensitization or after peanut sensitization and manifestation of the peanut-induced phenotype, blocked IgE-mediated alterations in peanut-induced histamine release, anaphylaxis scores, locomotor activity, and free IgE levels and protected animals from death caused by anaphylaxis. If this degree of persistent efficacy translates to human subjects, AAVrh.10anti-hIgE could be an effective 1-time preventative therapy for peanut allergy and possibly other severe, IgE-mediated allergies.

755. One-time Gene Therapy for Hereditary Angioedema

Hereditary angioedema (HAE) is a potentially life-threatening autosomal dominant deficiency affecting 1 in 50,000 individuals from all ethnic groups worldwide. More than 99% of HAE cases are caused by a deficiency in functional plasma C1 esterase inhibitor (C1E-INH, a serine protease inhibitor) due to mutations in the SERPING1 gene. Low plasma C1E-INH activity dysregulates the contact, complement, and fibrinolytic systems, resulting in unpredictable, recurrent submucosal edema of cutaneous tissues, gastrointestinal and respiratory tracts. If not treated in a timely manner, laryngeal edema can result in death by asphyxiation. Current HAE treatments consist of management of acute attacks, repeated prophylactic therapy with C1E-INH or long term prophylaxis with attenuated androgens, each complicated by a high economic burden, limited compliance, drug side effects and contraindications. To circumvent this challenge, we hypothesized that a one-time administration of an adeno-associated virus (AAV) gene transfer vector expressing the genetic sequence of C1 esterase-inhibitor (serotype 10 expressing the human CIE-INH coding sequence, AAVrh.10hC1EI) would provide sustained C1E-INH activity levels in plasma, sufficient to prevent angioedema episodes. To study the efficacy of AAVrh.10hC1EI, using CRISPR/Cas9 technology we created a novel C1E-INH deficient mouse model analogous to human HAE disease, and characterized the resulting SERPING1 gene mutations by genome sequencing. The heterozygous mouse model shares characteristics associated with HAE in humans including decreased C1E-INH and C4 levels in plasma and increased vascular permeability. Administration of AAVrh.10hC1EI to the heterozygous mice resulted in sustained human C1E levels above the predicted therapeutic levels. In order to demonstrate that the increased vascular permeability observed in heterozygote C1E-INH+/- mice was a direct result of C1E-INH deficiency, Evans blue dye was injected intravenously and extravasation of dye from the vasculature evaluated. Compared to wild type mice under baseline conditions, the C1EINH+/- mice had increased extravasation of the dye into the hind paws quantitated by optical absorbance at 600 nm. Strikingly, AAVrh10.hC1EI-treated (1011 gc) mice displayed a marked decrease in dye extravasation, whereas non-treated C1E-INH +/- mice had markedly increased dye extravasation. These results demonstrate that a single treatment with AAVrh.10hC1EI has the potential to provide long term protection from angioedema attacks in the affected population, representing a paradigm shift in current therapeutic approaches.

474. Consequences of Infusion Time on Efficiency of Intravenous Delivery of Vector Genomes to the Liver

Based on the knowledge that, when administered intravenously (IV), 90% of adeno-associated virus (AAV) gene transfer vectors are transferred to the liver, the IV route has become the standard for gene therapy applications directed toward genetic modification of liver cells. To achieve maximum transfer of vector genomes to the liver, most attention has been focused on AAV serotype and dose, but little focus has been directed toward whether differences could be achieved by varying the timing of IV vector administration. To assess this variable, we administered rh. 10 serotype AAV vectors intravenously to rodents using 2 variables: (1) single vs multiple administrations; and (2) length of time of infusion. To assess single vs multiple administration, mouse cohorts (C57Bl/6 males, n=5/group) were administered AAVrh.10hα1AT, encoding for human α1-antitrypsin (α1AT), IV at 1012 genome copies (gc)/kg, 4×1012 gc/kg and a 3rd cohort receiving the 1012 gc/kg dose on 4 consecutive days. Mice were bled at 4, 14, 28, and 112 days post-administration. Serum human αlAT levels showed the expected dose response. Mice administered 1012 gc/kg daily for 4 days had αlAT serum levels below those achieved by the single dose 4×1012 gc/kg group (day 112, p 0.1, for all time comparisons in each dosage group). We conclude that, at least in rodents, the use of multiple daily injections influences the expression patterns of the targeted gene; however, the rate of infusion does not appear to be a critical parameter for the design of intravenous infusion for targeting AAV vectors to liver.

301. AAV Capsid Engineering to Improve Transduction in Retina and Brain

Gene therapy vectors based on adeno-associated virus (AAV) are currently in clinical studies for numerous disease indications including Leber's congenital amaurosis, age-related macular degeneration, hemophilia, muscular dystrophy and Parkinson's disease. AAV vectors hold considerable promise as therapeutic agents; however there is potential to further improve the efficiency of AAV gene delivery and efficacy by making modifications to the AAV capsid. The AAV capsid can be engineered to incorporate mutations that alter its transduction activity, tropism, biodistribution and immunogenicity. We have constructed variant AAV vectors harboring a variety of capsid modifications including those that negate receptor binding and have tested these vectors in several tissues including the eye and brain. One variant, AAV2HBKO, is an AAV2 based vector containing mutations of critical amino acids known to be required for binding to its receptor, heparin sulfate proteoglycan. Interestingly, an AAV2HBKO vector delivering a secreted transgene, sFLT02, unexpectedly resulted in a 2-log increase in transduction compared to parental AAV2 when delivered subretinally to the mouse eye. Subretinal delivery of an AAV2HBKO vector expressing EGFP demonstrated that these capsid modifications resulted in an increase in photoreceptor transduction compared to the unmodified AAV2 vector. In contrast, the AAV2HBKO vector demonstrated a lack of transduction activity following intravitreal delivery to the mouse eye. In addition, we evaluated the transduction and tropism of AAV2HBKO in the mouse brain. In a head to head comparison with AAV2, the AAV2HBKO vector facilitated widespread striatal and cortical expression following an intrastriatal injection while AAV2-mediated expression was restricted to the site of injection. Similar to AAV2, the tropism of AAV2HBKO was primarily neuronal with little to no transduction of astrocytes or microglia. Biodistribution data suggests that this vector, when delivered systemically in the mouse, has significantly reduced liver transduction but a higher propensity to be delivered to skeletal muscle and heart compared to the wild-type AAV2 vector. We will present data evaluating the transduction activity, tropism and biodistribution of the AAV2HBKO variant. These studies illustrate the potential for improving the efficiency of AAV gene transfer via targeted capsid engineering.

80. Delayed Inflammatory Response to Intravitreal AAV Gene Transfer in Non-Human Primates

Introduction Adeno-associated virus (AAV) gene therapy in the eye via intravitreal injection has recently advanced into clinical trials. Here, we wanted to investigate the immunological response to AAV capsids within the vitreous of non-human primates. Methods Bilateral injections of AAV gene delivery were evaluated in five rhesus macaques. An intravitreal injection (IVT) was performed in one eye of the non-human primate, while a subretinal injection was performed in the contralateral eye. For the IVT eye, AAV2 or synthetic ancestral Anc80 capsid with GFP transgene was used at a concentration of 1E+11 particles. The surgical procedure for two animals was a normal IVT injection as performed in the clinic and the other two animals received an inner limiting membrane peel before viral injection. In the subretinal injection (SR) eye, animals were treated with AAV8 or 9, carrying the GFP or LacZ transgene at a concentration of 1E+10 or 1E+11 particles. Animals were monitored for 5/6 weeks before being sacrificed for histological analysis. Results In all eyes, inflammation in the first 2 weeks was mild or moderate and self-resolving. At weeks 3-5, all IVT treated eyes developed vitritis. None of the eyes injected subretinally developed vitritis. The most severe eye belonged to the IVT group, which developed hyphema and most of the IVT eyes developed anterior cataracts. Inflammation was well controlled with 4mg to 12mg of Triescence (triamcinolone acetonide) injected intravitrally. One animal was not given Triescence at the onset of inflammation (week 3), which became unmanageable within 48 hours, leading to the ultimate termination of the animal. Within the IVT group, similar amounts of late inflammation were seen between the intravitreal and peel/puddle eyes. Conclusion Our studies demonstrate that, in stark contrast to subretinal injection, an intravitreal injection of AAV expressing a non-self transgene has the potential to develop clinically significant inflammation at doses normally tolerated in the subretina. This inflammation can be treated with early administration of Triescence. Left untreated, the animal will develop severe immunological infiltration. These results warrant further study to determine the antigenic nature and inflammatory mechanism leading to the low inflammatory threshold to intravitreal AAV gene transfer.

Potential for cellular stress response to hepatic factor VIII expression from AAV vector

Hemophilia A and B are coagulation disorders resulting from the loss of functional coagulation factor VIII (FVIII) or factor IX proteins, respectively. Gene therapy for hemophilia with adeno-associated virus vectors has shown efficacy in hemophilia B patients. Although hemophilia A patients are more prevalent, the development of therapeutic adeno-associated virus vectors has been impeded by the size of the F8 cDNA and impaired secretion of FVIII protein. Further, it has been reported that over-expression of the FVIII protein induces endoplasmic reticulum stress and activates the unfolded protein response pathway both in vitro and in hepatocytes in vivo, presumably due to retention of misfolded FVIII protein within the endoplasmic reticulum. Engineering of the F8 transgene, including removal of the B domain (BDD-FVIII) and codon optimization, now allows for the generation of adeno-associated virus vectors capable of expressing therapeutic levels of FVIII. Here we sought to determine if the risks of inducing the unfolded protein response in murine hepatocytes extend to adeno-associated virus gene transfer. Although our data show a mild activation of unfolded protein response markers following F8 gene delivery at a certain vector dose in C57BL/6 mice, it was not augmented upon further elevated dosing, did not induce liver pathology or apoptosis, and did not impact FVIII immunogenicity.

AAV3 Capsid Is Superior for In Vivo Gene Transfer to Human Hepatocytes Compared to Serotypes 5 and 8 in a Mouse/Human Chimeric Model

Adeno-associated viral (AAV) gene transfer to the liver for the treatment of hemophilia B is currently being tested in multiple clinical trials and is also in the pipeline for hemophilia A. Current trials utilize AAV serotype 8, while other capsids including AAV5 will also be tested in hemophilia B patients in the near future. AAV8 shows superior transduction of murine liver and has directed long-term factor IX gene transfer in humans. However, efficacy was substantially lower in human than in murine liver. In order to better predict the performance of the various candidate capsids with liver tropism in humans, "humanized mice" harboring human hepatocytes have been adapted as a pre-clinical model of AAV liver gene transfer. Here, we utilized the liver chimeric FAH model. This model is based on the observation that adult immunodeficient fah-/- mice, which suffer from liver injury when cycled off the tyrosine catabolism blocker NTBC, support the proliferation of human hepatocytes. We used liver chimeric mice highly engrafted with fetal human hepatoblasts (human serum albumin levels 978-3737mcg/ml) or human pediatric hepatocytes (human serum albumin levels 388-3798mcg/ml). We produced AAV vectors expressing enhanced GFP reporter from a self-complementary genome. These included AAV5, AAV8, and also AAV3 (which in recent years has shown high transduction efficiency for human hepatocytes and human liver cancer cell lines in vitro and in murine xenograft models). Vectors were injected into the tail veins of humanized mice at a dose of 1x10^11 vector genomes/mouse. Livers were harvested 14 days later and analyzed for GFP expression in murine and human hepatocytes. Human cells were specifically identified by immunostain for FAH. We analyzed transduction efficiency by immunohistochemical stains of liver sections obtained from formalin fixed tissue (n=3 per vector). Transduced human FAH+GFP+ hepatocytes were visualized by confocal immunofluorescence microscopy followed by analysis with Velocity software. To further substantiate the results obtained by image analysis, we developed a flow cytometry-based method for detection of GFP-transduced FAH+ human hepatocytes and mCD81+ murine hepatocytes. Regardless of the method of analysis, AAV3 was the most efficient in transducing human hepatocytes, transducing 12-15% of human but only 3.5-5.5% of murine hepatocytes. AAV8 consistently transduced 5-6% of human hepatocytes while showing by far the highest efficiency for murine hepatocytes (on average 46%). Since the GFP signal in human hepatocytes transduced with AAV3 was higher than for AAV8 transduced cells, the difference in transduction efficiency of the human cells was likely greater in favor of AAV3 than the approximately 3-fold difference in percent GFP positivity. In contrast, AAV5 performed poorly, transducing only 0.1-0.3% of human hepatocytes and at best 0.8% of murine hepatocytes. This may in part also reflect the previously noted reduction in liver transduction when AAV5 is administered from a peripheral vein. Our model suggests that AAV5 is considerably less efficacious, in particular when given via a peripheral vein. In conclusion, AAV3-based vectors may improve liver-directed human gene therapy for hemophilia compared to the current prevailing AAV8 vector system. DisclosuresHerzog:Novo Nordisk: Research Funding; Spark Therapeutics: Patents & Royalties: Patent licenses. High:Spark Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties: AAV gene transfer technology. Srivastava:University of Florida: Patents & Royalties: AAV vector technology.

Hepatic AAV Gene Transfer of Cytoplasmic Transgene Induces Transgene Product-Specific T Cell Activation Initially in the Liver and Celiac Lymph Node Preceding Treg Induction

In several published studies, we have shown induction of immune tolerance to coagulations factors by hepatic gene transfer to animals with hemophilia. Tolerance induction is influenced by a number of complex factors, most notably T cell activation and induction of antigen-specific CD4+CD25+FoxP3+ regulatory T cells (Treg). We sought to better understand antigen presentation to CD4+ T cells and the dynamics of the resulting T cell response. To characterize the interaction of adeno-associated virus (AAV) antigen expression in the liver with immune cells, we used an AAV8 vector, which have a high tropism for murine liver, expressing cytoplasmic ovalbumin (AAV8-Cyto-Ova) from the EF1α promoter. Use of AAV8-Cyto-Ova allowed us to eliminate effects from systemic antigen delivery. Vector was injected into the tail vein of DO11.10-transgenic RAG-/- mice, which contain exclusively Ova-specific CD4+ T cells and lack Treg. AAV8-Cyto-Ova caused upregulation of the very early activation marker CD69 on the CD4+ T cells as early as 2 weeks after gene transfer, with induced Treg emerging at about 3 weeks. CD69+CD4+ T cells were first observed in greatest numbers in the liver and celiac lymph node (LN), one of the liver-draining LN. This T cell activation persisted for several weeks. To better define the sites of T cell activation, we used the compound FTY720, which is an agonist of sphingosine-1-phosphate receptors and prevents migration of lymphocytes but does not alter T cell function. Two weeks after AAV8-Cyto-Ova, FTY720 sequestered activated T cells mostly in the liver and celiac LN, when compared to other lymphoid organs, indicating that these are the initial sites of T cell activation. At the 3-week time point, there were fewer activated T cells in the liver and celiac LN in mice that received FTY720, while instead accumulating in the blood. Most likely, activated T cells were prevented from reentering the lymphoid organs from the circulation, where they were sequestered. We conclude that T cells are first activated by AAV8-Cyto-Ova in the liver and celiac LN after two weeks, where they subsequently egress into the circulation and re-enter lymphoid tissues, with many returning to the liver and celiac LN. FTY720 given at 2 weeks prevented the newly activated T cells from leaving the liver and celiac LN. These results strongly suggest that antigen presentation and CD4+ T cell activation occur first in the liver and celiac LN, beginning about 2 weeks after vector administration. Consistent with this conclusion, adoptively transferred Ova-specific CD4+ T cells proliferated first and to a much greater degree in the celiac LN of AAV8-Cyto-Ova transduced mice. Inactiviating Kupffer cells with gadolinium chloride significantly reduced antigen-specific proliferation, illustrating the requirement for professional resident liver antigen-presenting cells. Furthermore, we show that - in contrast to the AAV expression of secreted Ova - Treg are exclusively extrathymically induced after AAV8-Cyto-Ova vector administration. These Treg are found in high numbers in the blood after 2 weeks in mice given the FTY720 compound, suggesting that these peripherally induced Treg quickly enter the circulation. In conclusion, the liver and its draining celiac LN are key sites for antigen presentation and T cell activation in response to transgene expression directed by hepatic gene transfer. Presentation of antigen derived from a non-secreted transgene product induces FoxP3+ Treg that rapidly distribute through the circulation. DisclosuresHerzog:Novo Nordisk: Research Funding; Spark Therapeutics: Patents & Royalties: Patent licenses.