Coevolution of Adeno-associated Virus Capsid Antigenicity and Tropism through a Structure-Guided Approach.

Adeno-associated viruses (AAV) are composed of nonenveloped, icosahedral protein shells that can be adapted to package and deliver recombinant therapeutic DNA. Approaches to engineer recombinant capsids for gene therapy applications have focused on rational design or library-based approaches that can address one or two desirable attributes; however, there is an unmet need to comprehensively improve AAV vector properties. Such cannot be achieved by utilizing sequence data alone but requires harnessing the three-dimensional (3D) structural properties of AAV capsids. Here, we solve the structures of a natural AAV isolate complexed with antibodies using cryo-electron microscopy and harness this structural information to engineer AAV capsid libraries through saturation mutagenesis of different antigenic footprints. Each surface loop was evolved by infectious cycling in the presence of a helper adenovirus to yield a new AAV variant that then serves as a template for evolving the next surface loop. This stepwise process yielded a humanized AAV8 capsid (AAVhum.8) displaying nonnatural surface loops that simultaneously display tropism for human hepatocytes, increased gene transfer efficiency, and neutralizing antibody evasion. Specifically, AAVhum.8 can better evade neutralizing antisera from multiple species than AAV8. Further, AAVhum.8 displays robust transduction in a human liver xenograft mouse model with expanded tropism for both murine and human hepatocytes. This work supports the hypothesis that critical properties, such as AAV capsid antibody evasion and tropism, can be coevolved by combining rational design and library-based evolution for clinical gene therapy.IMPORTANCE Clinical gene therapy with recombinant AAV vectors has largely relied on natural capsid isolates. There is an unmet need to comprehensively improve AAV tissue tropism, transduction efficiency, and antibody evasion. Such cannot be achieved by utilizing capsid sequence data alone but requires harnessing the 3D structural properties of AAV capsids. Here, we combine rational design and library-based evolution to coevolve multiple, desirable properties onto AAV by harnessing 3D structural information.

Structure-guided AAV capsid evolution strategies for enhanced CNS gene delivery.

Over the past 5 years, our laboratory has systematically developed a structure-guided library approach to evolve new adeno-associated virus (AAV) capsids with altered tissue tropism, higher transduction efficiency and the ability to evade pre-existing humoral immunity. Here, we provide a detailed protocol describing two distinct evolution strategies using structurally divergent AAV serotypes as templates, exemplified by improving CNS gene transfer efficiency in vivo. We outline four major components of our strategy: (i) structure-guided design of AAV capsid libraries, (ii) AAV library production, (iii) library cycling in single versus multiple animal models, followed by (iv) evaluation of lead AAV vector candidates in vivo. The protocol spans ~95 d, excluding gene expression analysis in vivo, and can vary depending on user experience, resources and experimental design. A distinguishing attribute of the current protocol is the focus on providing biomedical researchers with 3D structural information to guide evolution of precise 'hotspots' on AAV capsids. Furthermore, the protocol outlines two distinct methods for AAV library evolution consisting of adenovirus-enabled infectious cycling in a single species and noninfectious cycling in a cross-species manner. Notably, our workflow can be seamlessly merged with other RNA transcript-based library strategies and tailored for tissue-specific capsid selection. Overall, the procedures outlined herein can be adapted to expand the AAV vector toolkit for genetic manipulation of animal models and development of human gene therapies.

The relationship between multimorbidity, obesity and functional impairment in older adults.

BACKGROUND: Declining mortality rates and an aging population have contributed to increasing rates of multimorbidity (MM) in the United States. MM is strongly associated with a decline in physical function. Obesity is an important risk factor for the development of MM, and its prevalence continues to rise. Our study aimed to evaluate the associations between obesity, MM, and rates of functional limitations in older adults. METHODS: We analyzed body mass index (BMI) and self-reported comorbidity data from 7261 individuals aged ≥60 years from the National Health and Nutrition Examination Surveys 2005-2014. Weight status was defined based on standard BMI categories. MM was defined as 2 or more comorbidities, while functional limitations were self-reported. Adjusted logistic regression quantified the association between standard BMI categories and MM. We also examined the difference in the prevalence of limitations between those with and without MM. RESULTS: The overall proportion of individuals with concomitant MM and obesity was 27.0%. Compared to a normal BMI, older adults with obesity had higher odds of MM (Prevalence odds ratio 1.79, 95% CI 1.49, 2.12). Overall, 67.5% of patients with MM also reported a functional limitation, with rates of functional limitation increasing with increasing BMI. When evaluating functional limitations in those with MM by BMI class, 90% of patients classified as severely obese (BMI ≥40 kg/m2 ) with MM also had a concomitant functional limitation. CONCLUSIONS: Compared to normal weight status, obesity is associated with an increased burden of MM and functional limitation among older adults. Our results underscore the importance of identifying and addressing obesity, MM, and functional limitation patterns and the need for evidence-based interventions that address all three conditions in this population.

Cross-species evolution of a highly potent AAV variant for therapeutic gene transfer and genome editing.

Recombinant adeno-associated viral (AAV) vectors are a promising gene delivery platform, but ongoing clinical trials continue to highlight a relatively narrow therapeutic window. Effective clinical translation is confounded, at least in part, by differences in AAV biology across animal species. Here, we tackle this challenge by sequentially evolving AAV capsid libraries in mice, pigs and macaques. We discover a highly potent, cross-species compatible variant (AAV.cc47) that shows improved attributes benchmarked against AAV serotype 9 as evidenced by robust reporter and therapeutic gene expression, Cre recombination and CRISPR genome editing in normal and diseased mouse models. Enhanced transduction efficiency of AAV.cc47 vectors is further corroborated in macaques and pigs, providing a strong rationale for potential clinical translation into human gene therapies. We envision that ccAAV vectors may not only improve predictive modeling in preclinical studies, but also clinical translatability by broadening the therapeutic window of AAV based gene therapies.

Engineering highly efficient backsplicing and translation of synthetic circRNAs.

Circular RNAs (circRNAs) are highly stable RNA molecules that are attractive templates for expression of therapeutic proteins and non-coding RNAs. In eukaryotes, circRNAs are primarily generated by the spliceosome through backsplicing. Here, we interrogate different molecular elements including intron type and length, Alu repeats, internal ribosome entry sites (IRESs), and exon length essential for circRNA formation and exploit this information to engineer robust backsplicing and circRNA expression. Specifically, we leverage the finding that the downstream intron can tolerate large inserts without affecting splicing to achieve tandem expression of backspliced circRNAs and tRNA intronic circRNAs from the same template. Further, truncation of selected intronic regions markedly increased circRNA formation in different cell types in vitro as well as AAV-mediated circRNA expression in cardiac and skeletal muscle tissue in vivo. We also observed that different IRES elements and exon length influenced circRNA expression and translation, revealing an exonic contribution to splicing, as evidenced by different RNA species produced. Taken together, these data provide new insight into improving the design and expression of synthetic circRNAs. When combined with AAV capsid and promoter technologies, the backsplicing introns and IRES elements constituting this modular platform significantly expand the gene expression toolkit.

Rescuing AAV gene transfer from neutralizing antibodies with an IgG-degrading enzyme.

Preexisting humoral immunity to recombinant adeno-associated virus (AAV) vectors restricts the treatable patient population and efficacy of human gene therapies. Approaches to clear neutralizing antibodies (NAbs), such as plasmapheresis and immunosuppression, are either ineffective or cause undesirable side effects. Here, we describe a clinically relevant strategy to rapidly and transiently degrade NAbs before AAV administration using an IgG-degrading enzyme (IdeZ). We demonstrate that recombinant IdeZ efficiently cleaved IgG in dog, monkey, and human antisera. Prophylactically administered IdeZ cleaved circulating human IgG in mice and prevented AAV neutralization in vivo. In macaques, a single intravenous dose of IdeZ rescued AAV transduction by transiently reversing seropositivity. Importantly, IdeZ efficiently cleaved NAbs and rescued AAV transduction in mice passively immunized with individual human donor sera representing a diverse population. Our antibody clearance approach presents a potentially new paradigm for expanding the prospective patient cohort and improving efficacy of AAV gene therapy.