Muscle-directed AAV gene therapy rescues the maple syrup urine disease phenotype in a mouse model.

Maple syrup urine disease (MSUD) is a rare, inherited metabolic disorder characterized by a dysfunctional mitochondrial enzyme complex, branched-chain alpha-keto acid dehydrogenase (BCKDH), which catabolizes branched-chain amino acids (BCAAs). Without functional BCKDH, BCAAs and their neurotoxic alpha-keto intermediates can accumulate in the blood and tissues. MSUD is currently incurable and treatment is limited to dietary restriction or liver transplantation, meaning there is a great need to develop new treatments for MSUD. We evaluated potential gene therapy applications for MSUD in the intermediate MSUD (iMSUD) mouse model, which harbors a mutation in the dihydrolipoamide branched-chain transacylase E2 (DBT) subunit of BCKDH. Systemic delivery of an adeno-associated virus (AAV) vector expressing DBT under control of the liver-specific TBG promoter to the liver did not sufficiently ameliorate all aspects of the disease phenotype. These findings necessitated an alternative therapeutic strategy. Muscle makes a larger contribution to BCAA metabolism than liver in humans, but a muscle-specific approach involving a muscle-specific promoter for DBT expression delivered via intramuscular (IM) administration only partially rescued the MSUD phenotype in mice. Combining the muscle-tropic AAV9 capsid with the ubiquitous CB7 promoter via IM or IV injection, however, substantially increased survival across all assessed doses. Additionally, near-normal serum BCAA levels were achieved and maintained in the mid- and high-dose cohorts throughout the study; this approach also protected these mice from a lethal high-protein diet challenge. Therefore, administration of a gene therapy vector that expresses in both muscle and liver may represent a viable approach to treating patients with MSUD.

TLR9 signaling mediates adaptive immunity following systemic AAV gene therapy.

An ongoing concern of in vivo gene therapy is adaptive immune responses against the protein product of a transgene, particularly for recessive diseases in which antigens are not presented to lymphocytes during central tolerance induction. Here we show that Toll-like receptor 9 (TLR9) signaling activates T cells against an epitope tagged mitochondria-targeted ornithine transcarbamylase (OTC) following the administration of a systemic adeno-associated virus (AAV) vector. Using a transgenic mouse model system, we demonstrate that TLR9 signaling extrinsic to T cells induces a robust cytotoxic T-cell response against the transgene and results in transgene expression loss. Overall, our results suggest that inflammation mediated by TLR9 signaling and the presence of high affinity transgene-specific T cells is important for the development of adaptive immune responses to transgene products following AAV gene therapy.

Alternative Start Sites Downstream of Non-Sense Mutations Drive Antigen Presentation and Tolerance Induction to C-Terminal Epitopes.

CTL responses to the transgene product remain an active area of concern for the gene therapy field. A patient's underlying genetic mutation may influence the qualitative nature of these potentially destructive T cell responses. Individuals with a mutation that introduces a premature termination codon (PTC) that prevents synthesis of the full-length peptide are considered more likely to mount a transgene-specific T cell response because of a lack of immune tolerance to C-terminal epitopes as a consequence of absent endogenous Ag presentation. In this article, we demonstrate that a human ornithine transcarbamylase gene containing various PTC-inducing non-sense mutations is able to generate and present epitopes downstream of the termination codon. Generation of these epitopes occurs primarily from alternative translation start sites downstream of the stop codon. Furthermore, we show that expression of these genes from adeno-associated virus vectors in C57BL/6 mice is able to induce peripheral tolerance to epitopes downstream of the PTC. These results suggest that, despite the lack of full-length endogenous protein, patients with PTC-inducing non-sense mutations may still present T cell epitopes downstream of the premature termination site that may render the subject tolerant to wild-type transgene products.

Adeno-associated viral gene therapy corrects a mouse model of argininosuccinic aciduria.

Argininosuccinic aciduria (ASA) is the second most common genetic disorder affecting the urea cycle. The disease is caused by deleterious mutations in the gene encoding argininosuccinate lyase (ASL); total loss of ASL activity results in severe neonatal onset of the disease, which is characterized by hyperammonemia within a few days of birth that can rapidly progress to coma and death. The long-term complications of ASA, such as hypertension and neurocognitive deficits, appear to be resistant to the current treatment options of dietary restriction, arginine supplementation, and nitrogen scavenging drugs. Treatment-resistant disease is currently being managed by orthotopic liver transplant, which shows variable improvement and requires lifetime immunosuppression. Here, we developed a gene therapy strategy for ASA aimed at alleviating the symptoms associated with urea cycle disruption by providing stable expression of ASL protein in the liver. We designed a codon-optimized human ASL gene packaged within adeno-associated virus serotype 8 (AAV8) as a vector for targeted delivery to the liver. To evaluate the therapeutic efficacy of this approach, we utilized a murine hypomorphic model of ASA. Neonatal administration of AAV8 via the temporal facial vein extended survival in ASA hypomorphic mice, although not to wild-type levels. Intravenous injection into adolescent hypomorphic mice led to increased survival and body weight and correction of metabolites associated with the disease. Our results demonstrate that AAV8 gene therapy is a viable approach for the treatment of ASA.

Treating Transthyretin Amyloidosis via Adeno-Associated Virus Vector Delivery of Meganucleases.

Transthyretin amyloidosis (ATTR) is a progressive and fatal disease caused by transthyretin (TTR) amyloid fibril accumulation in tissues, which disrupts organ function. As the TTR protein is primarily synthesized by the liver, liver transplantation can cure familial ATTR but is not an option for the predominant age-related wild-type ATTR. Approved treatment approaches include TTR stabilizers and an RNA-interference therapeutic, but these require regular re-administration. Gene editing could represent an effective one-time treatment. We evaluated adeno-associated virus (AAV) vector-delivered, gene-editing meganucleases to reduce TTR levels. We used engineered meganucleases targeting two different sites within the TTR gene. AAV vectors expressing TTR meganuclease transgenes were first tested in immunodeficient mice expressing the human TTR sequence delivered using an AAV vector and then against the endogenous TTR gene in rhesus macaques. Following a dose of 3 × 1013 genome copies per kilogram, we detected on-target editing efficiency of up to 45% insertions and deletions (indels) in the TTR genomic DNA locus and >80% indels in TTR RNA, with a concomitant decrease in serum TTR levels of >95% in macaques. The significant reduction in serum TTR levels following TTR gene editing indicates that this approach could be an effective treatment for ATTR.

A Gene Therapy Approach to Improve Copper Metabolism and Prevent Liver Damage in a Mouse Model of Wilson Disease.

Wilson disease (WD), an autosomal recessive disease caused by mutations in a copper-transporting P-type ATPase (Atp7b), causes severe liver damage. This disease is currently treated with the lifelong use of copper chelation therapy, which has side effects and does not fix copper metabolism. Here, we thoroughly characterized a mouse model of WD, the toxic milk mouse, and used the model to test a gene therapy approach for treating WD. WD mice accumulated copper in the liver from birth; severe copper accumulation and concurrent liver disease were evident by 2 months of age. Intravenously administering an adeno-associated viral (AAV) 8 vector expressing a codon-optimized version of the human ATP7B transgene into 2-month-old WD mice significantly decreased liver copper levels compared with age-matched, uninjected, WD mice. We also observed a significant dose-dependent decrease in liver disease. Male mice injected with 1011 genome copies of AAV8 vector showed only mild histopathological findings with a complete lack of liver fibrosis. Therefore, we conclude that administering gene therapy at the early stages of disease onset is a promising approach for reducing liver damage and correcting copper metabolism in WD.

CpG-depleted adeno-associated virus vectors evade immune detection.

Due to their efficient transduction potential, adeno-associated virus (AAV) vectors are leading candidates for gene therapy in skeletal muscle diseases. However, immune responses toward the vector or transgene product have been observed in preclinical and clinical studies. TLR9 has been implicated in promoting AAV-directed immune responses, but vectors have not been developed to circumvent this barrier. To assess the requirement of TLR9 in promoting immunity toward AAV-associated antigens following skeletal muscle gene transfer in mice, we compared immunological responses in WT and Tlr9-deficient mice that received an AAV vector with an immunogenic capsid, AAVrh32.33. In Tlr9-deficient mice, IFN-γ T cell responses toward capsid and transgene antigen were suppressed, resulting in minimal cellular infiltrate and stable transgene expression in target muscles. These findings suggest that AAV-directed immune responses may be circumvented by depleting the ligand for TLR9 (CpG sequences) from the vector genome. Indeed, we found that CpG-depleted AAVrh32.33 vectors could establish persistent transgene expression, evade immunity, and minimize infiltration of effector cells. Thus, CpG-depleted AAV vectors could improve outcome of clinical trials of gene therapy for skeletal muscle disease.