Adeno-associated virus-mediated trastuzumab delivery to the central nervous system for human epidermal growth factor receptor 2+ brain metastasis.

Trastuzumab improves overall survival for HER2+ breast cancer, but its short half-life in the cerebrospinal fluid (~2-4 days) and delivery limitations restrict the ability to target HER2+ central nervous system (CNS) disease. We developed an adeno-associated virus (AAV) vector expressing a codon-optimized, ubiquitin C (UbC)-promoter-driven trastuzumab sequence (AAV9.UbC.trastuzumab) for intrathecal administration. Transgene expression was evaluated in adult Rag1 knockout mice and rhesus nonhuman primates (NHPs) after a single intracerebroventricular (ICV) or intra-cisterna magna (ICM) AAV9.UbC.trastuzumab injection, respectively, using real-time PCR, ELISA, Western blot, in situ hybridization, single-nucleus RNA sequencing, and liquid chromatography-mass spectrometry; antitumor efficacy was evaluated in brain xenografts using HER2+ breast cancer cell lines (BT-474, MDA-MB-453). Transgene expression was detected in brain homogenates of Rag1 knockout mice following a single ICV injection of AAV9.UbC.trastuzumab (1 × 1011 vector genome copies [GC]/mouse) and tumor progression was inhibited in xenograft models of breast-to-brain metastasis. In NHPs, ICM delivery of AAV9.UbC.trastuzumab (3 × 1013 GC/animal) was well tolerated (36-37 days in-life) and resulted in transgene expression in CNS tissues and cerebrospinal fluid at levels sufficient to induce complete tumor remission in MDA-MB-453 brain xenografts. With AAV9's proven clinical safety record, this gene therapy may represent a viable approach for targeting HER2 + CNS malignancies.

Magnetic Resonance Imaging-Guided Frameless Stereotactic Injections of the Bilateral Cerebellar Dentate Nuclei in Nonhuman Primates: Technical Note.

BACKGROUND AND OBJECTIVES: Nonhuman primates (NHPs) are important preclinical models for evaluating therapeutics because of their anatomophysiological similarities to humans, and can be especially useful for testing new delivery targets. With the growing promise of cell and gene therapies for the treatment of neurological diseases, it is important to ensure the accurate and safe delivery of these agents to target structures in the brain. However, a standard guideline or method has not been developed for stereotactic targeting in NHPs. In this article, we describe the safe use of a magnetic resonance imaging-guided frameless stereotactic system to target bilateral cerebellar dentate nuclei for accurate, real-time delivery of viral vector in NHPs. METHODS: Seventeen rhesus macaques (Macaca mulatta) underwent stereotactic surgery under real-time MRI guidance using the ClearPoint® system. Bilateral cerebellar dentate nuclei were targeted through a single parietal entry point with a transtentorial approach. Fifty microliters of contrast-impregnated infusate was delivered to each dentate nucleus, and adjustments were made as necessary according to real-time MRI monitoring of delivery. Perioperative clinical outcomes and postoperative volumes of distribution were recorded. RESULTS: All macaques underwent bilateral surgery successfully. Superficial pin site infection occurred in 4/17 (23.5%) subjects, which resolved with antibiotics. Two episodes of transient neurological deficit (anisocoria and unilateral weakness) were recorded, which did not require additional postoperative treatment and resolved over time. Volume of distribution of infusate achieved satisfactory coverage of target dentate nuclei, and only 1 incidence (2.9%) of cerebrospinal fluid penetration was recorded. Mean volume of distribution was 161.22 ± 39.61 mm3 (left, 173.65 ± 48.29; right, 148.80 ± 23.98). CONCLUSION: MRI-guided frameless stereotactic injection of bilateral cerebellar dentate nuclei in NHPs is safe and feasible. The use of this technique enables real-time modification of the surgical plan to achieve adequate target coverage and can be readily translated to clinical use.

High-dose systemic adeno-associated virus vector administration causes liver and sinusoidal endothelial cell injury.

We analyzed retrospective data from toxicology studies involving administration of high doses of adeno-associated virus expressing different therapeutic transgenes to 21 cynomolgus and 15 rhesus macaques. We also conducted prospective studies to investigate acute toxicity following high-dose systemic administration of enhanced green fluorescent protein-expressing adeno-associated virus to 10 rhesus macaques. Toxicity was characterized by transaminitis, thrombocytopenia, and alternative complement pathway activation that peaked on post-administration day 3. Although most animals recovered, some developed ascites, generalized edema, hyperbilirubinemia, and/or coagulopathy that prompted unscheduled euthanasia. Study endpoint livers from animals that recovered and from unscheduled necropsies of those that succumbed to toxicity were analyzed via hypothesis-driven histopathology and unbiased single-nucleus RNA sequencing. All liver cell types expressed high transgene transcript levels at early unscheduled timepoints that subsequently decreased. Thrombocytopenia coincided with sinusoidal platelet microthrombi and sinusoidal endothelial injury identified via immunohistology and single-nucleus RNA sequencing. Acute toxicity, sinusoidal injury, and liver platelet sequestration were similarly observed with therapeutic transgenes and enhanced green fluorescent protein at doses ≥1 × 1014 GC/kg, suggesting it was the consequence of high-dose systemic adeno-associated virus administration, not green fluorescent protein toxicity. These findings highlight a potential toxic effect of high-dose intravenous adeno-associated virus on nonhuman primate liver microvasculature.

In situ combinatorial synthesis of degradable branched lipidoids for systemic delivery of mRNA therapeutics and gene editors.

The ionizable lipidoid is a key component of lipid nanoparticles (LNPs). Degradable lipidoids containing extended alkyl branches have received tremendous attention, yet their optimization and investigation are underappreciated. Here, we devise an in situ construction method for the combinatorial synthesis of degradable branched (DB) lipidoids. We find that appending branch tails to inefficacious lipidoids via degradable linkers boosts mRNA delivery efficiency up to three orders of magnitude. Combinatorial screening and systematic investigation of two libraries of DB-lipidoids reveal important structural criteria that govern their in vivo potency. The lead DB-LNP demonstrates robust delivery of mRNA therapeutics and gene editors into the liver. In a diet-induced obese mouse model, we show that repeated administration of DB-LNP encapsulating mRNA encoding human fibroblast growth factor 21 alleviates obesity and fatty liver. Together, we offer a construction strategy for high-throughput and cost-efficient synthesis of DB-lipidoids. This study provides insights into branched lipidoids for efficient mRNA delivery.

Early prediction of end-stage kidney disease using electronic health record data: a machine learning approach with a 2-year horizon.

Objectives: In the United States, end-stage kidney disease (ESKD) is responsible for high mortality and significant healthcare costs, with the number of cases sharply increasing in the past 2 decades. In this study, we aimed to reduce these impacts by developing an ESKD model for predicting its occurrence in a 2-year period. Materials and Methods: We developed a machine learning (ML) pipeline to test different models for the prediction of ESKD. The electronic health record was used to capture several kidney disease-related variables. Various imputation methods, feature selection, and sampling approaches were tested. We compared the performance of multiple ML models using area under the ROC curve (AUCROC), area under the Precision-Recall curve (PR-AUC), and Brier scores for discrimination, precision, and calibration, respectively. Explainability methods were applied to the final model. Results: Our best model was a gradient-boosting machine with feature selection and imputation methods as additional components. The model exhibited an AUCROC of 0.97, a PR-AUC of 0.33, and a Brier score of 0.002 on a holdout test set. A chart review analysis by expert physicians indicated clinical utility. Discussion and Conclusion: An ESKD prediction model can identify individuals at risk for ESKD and has been successfully deployed within our health system.

Integrated vector genomes may contribute to long-term expression in primate liver after AAV administration.

The development of liver-based adeno-associated virus (AAV) gene therapies is facing concerns about limited efficiency and durability of transgene expression. We evaluated nonhuman primates following intravenous dosing of AAV8 and AAVrh10 vectors for over 2 years to better define the mechanism(s) of transduction that affect performance. High transduction of non-immunogenic transgenes was achieved, although expression declined over the first 90 days to reach a lower but stable steady state. More than 10% of hepatocytes contained single nuclear domains of vector DNA that persisted despite the loss of transgene expression. Greater reductions in vector DNA and RNA were observed with immunogenic transgenes. Genomic integration of vector sequences, including complex concatemeric structures, were detected in 1 out of 100 cells at broadly distributed loci that were not in proximity to genes associated with hepatocellular carcinoma. Our studies suggest that AAV-mediated transgene expression in primate hepatocytes occurs in two phases: high but short-lived expression from episomal genomes, followed by much lower but stable expression, likely from integrated vectors.

Prevalent and Disseminated Recombinant and Wild-Type Adeno-Associated Virus Integration in Macaques and Humans.

Integration of naturally occurring adeno-associated viruses (AAV; wild-type AAV [wtAAV]) and those used in gene therapy (recombinant AAV [rAAV]) into host genomic DNA has been documented for over two decades. Results from mouse and dog studies have raised concerns of insertional mutagenesis and clonal expansion following AAV exposure, particularly in the context of gene therapy. This study aimed to characterize the genomic location, abundance, and expansion of wtAAV and rAAV integrations in macaque and human genomes. Using an unbiased, next-generation sequencing-based approach, we identified the genome-wide integration loci in tissue samples (primarily liver) in 168 nonhuman primates (NHPs) and 85 humans naïve to rAAV exposure and 86 NHPs treated with rAAV in preclinical studies. Our results suggest that rAAV and wtAAV integrations exhibit similar, broad distribution patterns across species, with a higher frequency in genomic regions highly vulnerable to DNA damage or close to highly transcribed genes. rAAV exhibited a higher abundance of unique integration loci, whereas wtAAV integration loci were associated with greater clonal expansion. This expansive and detailed characterization of AAV integration in NHPs and humans provides key translational insights, with important implications for the safety of rAAV as a gene therapy vector.

Quantification of human mature frataxin protein expression in nonhuman primate hearts after gene therapy.

Deficiency in human mature frataxin (hFXN-M) protein is responsible for the devastating neurodegenerative and cardiodegenerative disease of Friedreich's ataxia (FRDA). It results primarily through epigenetic silencing of the FXN gene by GAA triplet repeats on intron 1 of both alleles. GAA repeat lengths are most commonly between 600 and 1200 but can reach 1700. A subset of approximately 3% of FRDA patients have GAA repeats on one allele and a mutation on the other. FRDA patients die most commonly in their 30s from heart disease. Therefore, increasing expression of heart hFXN-M using gene therapy offers a way to prevent early mortality in FRDA. We used rhesus macaque monkeys to test the pharmacology of an adeno-associated virus (AAV)hu68.CB7.hFXN therapy. The advantage of using non-human primates for hFXN-M gene therapy studies is that hFXN-M and monkey FXN-M (mFXN-M) are 98.5% identical, which limits potential immunologic side-effects. However, this presented a formidable bioanalytical challenge in quantification of proteins with almost identical sequences. This could be overcome by the development of a species-specific quantitative mass spectrometry-based method, which has revealed for the first time, robust transgene-specific human protein expression in monkey heart tissue. The dose response is non-linear resulting in a ten-fold increase in monkey heart hFXN-M protein expression with only a three-fold increase in dose of the vector.

Adeno-associated virus-vectored erythropoietin gene therapy for anemia in cats with chronic kidney disease.

BACKGROUND: A treatment of chronic kidney disease (CKD)-associated anemia in cats is needed. SB-001 is an adeno-associated virus-vectored (AAV)-based gene therapeutic agent that is administered intramuscularly, causing the expression of feline erythropoietin. HYPOTHESIS/OBJECTIVE: We hypothesized that SB-001 injection would lead to a sustained increase in PCV in cats with CKD-associated anemia. ANIMALS: Twenty-three cats with International Renal Interest Society (IRIS) Stage 2 to 4 CKD-associated anemia were enrolled at 4 veterinary clinics. METHODS: In a prospective clinical trial, cats were treated with 1 of 3 regimens of SB-001 (Lo 1.2 × 109 genome copies [GCs] on Day 0; Lo ± Hi [supplemental 2nd dose of 3.65 × 109 GC on Day 42]; Hi 3.65 × 109 GC IM on Day 0) and followed for 70 days. RESULTS: A response to SB-001 at any time between Day 28 and Day 70 was seen in 86% (95% confidence interval 65, 97%) of all cats. There was a significant (P < .003) increase in PCV from Day 0 to Day 28 (mean increase 6 ± 6 percentage points [pp]; n = 21), Day 42 (8 ± 9 pp; n = 21), Day 56 (10 ± 11 pp; n = 17), and Day 70 (13 ± 14 pp, n = 14). Twelve cats were hypertensive at baseline, 4 of which developed encephalopathy during the study. An additional 6 cats became hypertensive during the study. CONCLUSIONS AND CLINICAL IMPORTANCE: Results of this study suggest that SB-001 therapy represents a suitable single injection treatment that can address nonregenerative anemia in cats with CKD. It was generally well tolerated; however, hypertension and encephalopathy developed in some cats as previously described in association with erythropoiesis-stimulating agent therapy.

Prednisolone and rapamycin reduce the plasma cell gene signature and may improve AAV gene therapy in cynomolgus macaques.

Adeno-associated virus (AAV) vector gene therapy is a promising approach to treat rare genetic diseases; however, an ongoing challenge is how to best modulate host immunity to improve transduction efficiency and therapeutic outcomes. This report presents two studies characterizing multiple prophylactic immunosuppression regimens in male cynomolgus macaques receiving an AAVrh10 gene therapy vector expressing human coagulation factor VIII (hFVIII). In study 1, no immunosuppression was compared with prednisolone, rapamycin (or sirolimus), rapamycin and cyclosporin A in combination, and cyclosporin A and azathioprine in combination. Prednisolone alone demonstrated higher mean peripheral blood hFVIII expression; however, this was not sustained upon taper. Anti-capsid and anti-hFVIII antibody responses were robust, and vector genomes and transgene mRNA levels were similar to no immunosuppression at necropsy. Study 2 compared no immunosuppression with prednisolone alone or in combination with rapamycin or methotrexate. The prednisolone/rapamycin group demonstrated an increase in mean hFVIII expression and a mean delay in anti-capsid IgG development until after rapamycin taper. Additionally, a significant reduction in the plasma cell gene signature was observed with prednisolone/rapamycin, suggesting that rapamycin's tolerogenic effects may include plasma cell differentiation blockade. Immunosuppression with prednisolone and rapamycin in combination could improve therapeutic outcomes in AAV vector gene therapy.

Positron Emission Tomography Quantitative Assessment of Off-Target Whole-Body Biodistribution of I-124-Labeled Adeno-Associated Virus Capsids Administered to Cerebral Spinal Fluid.

Based on studies in experimental animals demonstrating that administration of adeno-associated virus (AAV) vectors to the cerebrospinal fluid (CSF) is an effective route to transfer genes to the nervous system, there are increasing number of clinical trials using the CSF route to treat nervous system disorders. With the knowledge that the CSF turns over four to five times daily, and evidence in experimental animals that at least some of CSF administered AAV vectors are distributed to systemic organs, we asked: with AAV administration to the CSF, what fraction of the total dose remains in the nervous system and what fraction goes off target and is delivered systemically? To quantify the biodistribution of AAV capsids immediately after administration, we covalently labeled AAV capsids with iodine 124 (I-124), a cyclotron generated positron emitter, enabling quantitative positron emission tomography scanning of capsid distribution for up to 96 h after AAV vector administration. We assessed the biodistribution to nonhuman primates of I-124-labeled capsids from different AAV clades, including 9 (clade F), rh.10 (E), PHP.eB (F), hu68 (F), and rh91(A). The analysis demonstrated that 60-90% of AAV vectors administered to the CSF through either the intracisternal or intrathecal (lumbar) routes distributed systemically to major organs. These observations have potentially significant clinical implications regarding accuracy of AAV vector dosing to the nervous system, evoking systemic immunity at levels similar to that with systemic administration, and potential toxicity of genes designed to treat nervous system disorders being expressed in non-nervous system organs. Based on these data, individuals in clinical trials using AAV vectors administered to the CSF should be monitored for systemic as well as nervous system adverse events and CNS dosing considerations should account for a significant AAV systemic distribution.

Significant Differences in Capsid Properties and Potency Between Adeno-Associated Virus Vectors Produced in Sf9 and HEK293 Cells.

For successful vector-based gene therapy manufacturing, the selected adeno-associated virus (AAV) vector production system must produce vector at sufficient scale. However, concerns have arisen regarding the quality of vector produced using different systems. In this study, we compared AAV serotypes 1, 8, and 9 produced by two different systems (Sf9/baculovirus and HEK293/transfection) and purified by two separate processes. We evaluated capsid properties, including protein composition, post-translational modification, particle content profiles, and in vitro and in vivo vector potency. Vectors produced in the Sf9/baculovirus system displayed reduced incorporation of viral protein 1 and 2 into the capsid, increased capsid protein deamidation, increased empty and partially packaged particles in vector preparations, and an overall reduced potency. The differences observed were largely independent of the harvest method and purification process. These findings illustrate the need for careful consideration when choosing an AAV vector production system for clinical production.

Vector Affinity and Receptor Distribution Define Tissue-Specific Targeting in an Engineered AAV Capsid.

Unbiased in vivo selections of diverse capsid libraries can yield engineered capsids that overcome gene therapy delivery challenges like traversing the blood-brain barrier (BBB), but little is known about the parameters of capsid-receptor interactions that govern their improved activity. This hampers broader efforts in precision capsid engineering and is a practical impediment to ensuring the translatability of capsid properties between preclinical animal models and human clinical trials. In this work, we utilize the adeno-associated virus (AAV)-PHP.B-Ly6a model system to better understand the targeted delivery and BBB penetration properties of AAV vectors. This model offers a defined capsid-receptor pair that can be used to systematically define relationships between target receptor affinity and in vivo activity of engineered AAV vectors. Here, we report a high-throughput method for quantifying capsid-receptor affinity and demonstrate that direct binding assays can be used to organize a vector library into families with varied affinity for their target receptor. Our data indicate that efficient central nervous system transduction requires high levels of target receptor expression at the BBB, but it is not a requirement for receptor expression to be limited to the target tissue. We observed that enhanced receptor affinity leads to reduced transduction of off-target tissues but can negatively impact on-target cellular transduction and penetration of endothelial barriers. Together, this work provides a set of tools for defining vector-receptor affinities and demonstrates how receptor expression and affinity interact to impact the performance of engineered AAV vectors in targeting the central nervous system. IMPORTANCE Novel methods for measuring adeno-associated virus (AAV)-receptor affinities, especially in relation to vector performance in vivo, would be useful to capsid engineers as they develop AAV vectors for gene therapy applications and characterize their interactions with native or engineered receptors. Here, we use the AAV-PHP.B-Ly6a model system to assess the impact of receptor affinity on the systemic delivery and endothelial penetration properties of AAV-PHP.B vectors. We discuss how receptor affinity analysis can be used to isolate vectors with optimized properties, improve the interpretation of library selections, and ultimately translate vector activities between preclinical animal models and humans.

Immune transgene-dependent myocarditis in macaques after systemic administration of adeno-associated virus expressing human acid alpha-glucosidase.

Immune responses to human non-self transgenes can present challenges in preclinical studies of adeno-associated virus (AAV) gene therapy candidates in nonhuman primates. Although anti-transgene immune responses are usually mild and non-adverse, they can confound pharmacological readouts and complicate translation of results between species. We developed a gene therapy candidate for Pompe disease consisting of AAVhu68, a clade F AAV closely related to AAV9, that expresses an engineered human acid-alpha glucosidase (hGAA) tagged with an insulin-like growth factor 2 variant (vIGF2) peptide for enhanced cell uptake. Rhesus macaques were administered an intravenous dose of 1x1013 genome copies (GC)/kg, 5x1013 GC/kg, or 1 x 1014 GC/kg of AAVhu68.vIGF2.hGAA. Some unusually severe adaptive immune responses to hGAA presented, albeit with a high degree of variability between animals. Anti-hGAA responses ranged from absent to severe cytotoxic T-cell-mediated myocarditis with elevated troponin I levels. Cardiac toxicity was not dose dependent and affected five out of eleven animals. Upon further investigation, we identified an association between toxicity and a major histocompatibility complex class I haplotype (Mamu-A002.01) in three of these animals. An immunodominant peptide located in the C-terminal region of hGAA was subsequently identified via enzyme-linked immunospot epitope mapping. Another notable observation in this preclinical safety study cohort pertained to the achievement of robust and safe gene transfer upon intravenous administration of 5x1013 GC/kg in one animal with a low pre-existing neutralizing anti-capsid antibodies titer (1:20). Collectively, these findings may have significant implications for gene therapy inclusion criteria.

Lipid nanoparticle-encapsulated mRNA therapy corrects serum total bilirubin level in Crigler-Najjar syndrome mouse model.

Crigler-Najjar syndrome is a rare disorder of bilirubin metabolism caused by uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1) mutations characterized by hyperbilirubinemia and jaundice. No cure currently exists; treatment options are limited to phototherapy, whose effectiveness diminishes over time, and liver transplantation. Here, we evaluated the therapeutic potential of systemically administered, lipid nanoparticle-encapsulated human UGT1A1 (hUGT1A1) mRNA therapy in a Crigler-Najjar mouse model. Ugt1 knockout mice were rescued from lethal post-natal hyperbilirubinemia by phototherapy. These adult Ugt1 knockout mice were then administered a single lipid nanoparticle-encapsulated hUGT1A1 mRNA dose. Within 24 h, serum total bilirubin levels decreased from 15 mg/dL (256 µmol/L) to <0.5 mg/dL (9 µmol/L), i.e., slightly above wild-type levels. This reduction was sustained for 2 weeks before bilirubin levels rose and returned to pre-treatment levels by day 42 post-administration. Sustained reductions in total bilirubin levels were achieved by repeated administration of the mRNA product in a frequency-dependent manner. We were also able to rescue the neonatal lethality phenotype seen in Ugt1 knockout mice with a single lipid nanoparticle dose, which suggests that this may be a treatment modality appropriate for metabolic crisis situations. Therefore, lipid nanoparticle-encapsulated hUGT1A1 mRNA may represent a potential treatment for Crigler-Najjar syndrome.

Neonatal Fc Receptor Inhibition Enables Adeno-Associated Virus Gene Therapy Despite Pre-Existing Humoral Immunity.

Advances in adeno-associated virus (AAV)-based gene therapy are transforming our ability to treat rare genetic disorders and address other unmet medical needs. However, the natural prevalence of anti-AAV neutralizing antibodies (NAbs) in humans currently limits the population who can benefit from AAV-based gene therapies. Neonatal Fc receptor (FcRn) plays an essential role in the long half-life of IgG, a key NAb. Researchers have developed several FcRn-inhibiting monoclonal antibodies to treat autoimmune diseases, as inhibiting the interaction between FcRn and IgG Fc can reduce circulating IgG levels to 20-30% of the baseline. We evaluated the utility of one such monoclonal antibody, M281, to reduce pre-existing NAb levels and to permit gene delivery to the liver and heart via systemic AAV gene therapy in mice and nonhuman primates. M281 successfully reduced NAb titers along with total IgG levels; it also enhanced gene delivery to the liver and other organs after intravenous administration of AAV in NAb-positive animals. These results indicate that mitigating pre-existing humoral immunity via disruption of the FcRn-IgG interaction may make AAV-based gene therapies effective in NAb-positive patients.

Intravenous immunoglobulin prevents peripheral liver transduction of intrathecally delivered AAV vectors.

Gene therapy using neurotropic adeno-associated virus vectors represents an emerging solution for genetic disorders affecting the central nervous system. The first approved central nervous system-targeting adeno-associated virus gene therapy, Zolgensma®, for treating spinal muscular atrophy is administered intravenously at high doses that cause liver-associated adverse events in 20%-30% of patients. Intrathecal routes of vector administration, such as the intra-cisterna magna route, provide efficient gene transduction to central nervous system cells while reducing off-target liver transduction. However, significant levels of liver transduction often occur upon intra-cisterna magna vector delivery in preclinical studies. Using vectors expressing monoclonal antibody transgenes, we examined whether passive transfer of adeno-associated virus-neutralizing antibodies as intravenous immunoglobulin before intrathecal adeno-associated virus delivery improved the safety of viral gene therapy targeting the central nervous system in mice and nonhuman primates. We used intracerebroventricular and intra-cisterna magna routes for vector administration to mice and nonhuman primates, respectively, and evaluated transgene expression and vector genome distribution. Our data indicate that pretreatment with intravenous immunoglobulin significantly reduced gene transduction to the liver and other peripheral organs but not to the central nervous system in both species. With further refinement, this method may improve the safety of adeno-associated virus-based, central nervous system-targeting gene therapies in clinical settings.

Erratum: Intravenous immunoglobulin prevents peripheral liver transduction of intrathecally delivered AAV vectors.

[This corrects the article DOI: 10.1016/j.omtm.2022.09.017.].

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.