Let’s switch on AAV!

Our lab is mainly focused on RNA switch engineering and regulatable gene therapy development. Adeno-associated virus (AAV) vector-mediated in vivo somatic cell gene therapies, which utilize a single dose AAV to provide integration-independent persistent transgene expression for 10 years or more, have shown great therapeutic potential for treating a wide range of human diseases. However, safer and broader applications of AAV gene therapy are in part limited by the lack of useful molecular tools for precise control of therapeutic transgene expression. RNA switches, structured small noncoding RNA domains that control gene expression independent of any protein factors, represent a class of very promising tools for transgene regulation. A body of our prior studies have moved artificial RNA switches from working efficiently in vitro to now functioning with wide dynamic ranges in animals. These studies also established a strong proof of concept that RNA switches could enable safer and more effective applications and expand the use of AAV gene therapies to broader disease indications. Learn more about regulatable gene therapy here.

 We are now very interested in the following three directions:

1. Tool development: engineering parts (novel RNA switches, other RNA-based tools, therapeutic payloads) for the development of AAV- or mRNA-based in vivo gene therapies.
   
   
2. Mechanistic studies: investigating how the in-house developed RNA switches work in mammalian cells.
   
   
3. Gene therapy applications: developing RNA switch-regulated tunable and/or terminable AAV gene-addition/gene-editing therapies or mRNA-based therapies for fatal genetic diseases (e.g. Duchenne muscular dystrophy, Huntington’s disease), metabolic disorders (e.g. leptin deficiency, NASH), and infectious diseases (e.g. COVID-19).

 We are always interested in possible collaborations with researchers who focus on the biology of specific diseases or novel therapeutic targets where our regulatable gene therapy technology might be applied.

Ongoing projects in the lab

RNA switch-regulated, all-in-one AAV-CRISPR/Cas systems for in vivo inducible gene editing therapies

CRISPR-Cas-mediated in vivo somatic cell gene editing has enormous potential for treating a range of human diseases. Recombinant adeno-associated virus (AAV) vector is an efficient delivery vehicle for in vivo gene editing in diverse tissues. However, a single dose AAV could provide sustained transgene expression for 10 years or more in humans, which could lead to persistent off-target editing, long-lasting expression of an immunogenic Cas effector protein, and high level of double-strand break-associated integration of AAV vector DNA into chromosomal DNA. These potential safety issues significantly increase the risk, undercut the efficacy, and thus limit the application of this approach. An inducible CRISPR-Cas expression system has the potential to address these safety issues.

We have previously developed a self-cleaving ribozyme-based RNA on switch system that efficiently regulates AAV transgene expression with up to 200-fold dynamic range in mouse (Zhong et al. Nature Biotechnology. 2020). In this system, an in-house optimized, self-cleaving hammerhead ribozyme T3H38 is built in the 3ʹ-untranslated region (UTR) of an AAV transgene and keeps the transgene in the "off" state through self-cleavage-mediated disruption of transgene mRNA. A steric-blocking antisense oligonucleotide (ASO) v-M8, which is complementary to the ribozyme and provided in trans, could block the ribozyme’s self-cleavage and thus switch on and tune the expression of the transgene. Utilizing this T3H38/v-M8 RNA switch system, we achieved v-M8 dose-dependent control of AAV transgenes in mice. Based on this T3H38/v-M8 RNA switch system, we have recently developed a multilayer-regulated inducible CRISPR/Cas system that can be delivered by a single AAV vector. We demonstrated in cell culture as well as in mice that the system had minimal leaky editing activity before induction, and can be efficiently switched on by a single v-M8 morpholino inducer. This RNA switch-regulated all-in-one AAV-CRISPR/Cas system lays the foundation for developing safer and effective in vivo gene-editing therapies for a range of disorders of muscle or CNS origin. More details will be shared here as soon as we can.

RNA switch-regulated AAV systems for long-term and tunable expression of short-lived therapeutic protein hormones

Long-term delivery of short-lived protein hormones are generally desirable for the treatment of diverse diseases ranging from chronic anemia, to diabetes, to non-alcoholic steatohepatitis (NASH). AAV-delivered in vivo gene therapy can turn transduced somatic cells (e.g. skeletal muscle cells) into a bio-factory to support long-lasting production of secreted therapeutic proteins. RNA switch-regulated protein hormone gene therapy, which utilizes AAV transduced somatic cells to continuously produce and secrete protein hormones in their very native forms into the bloodstream, is therefore an attractive and generalizable approach to long-term delivery of short-lived protein hormones for a wide range of diseases that require chronic use of hormonal-based pharmacotherapies.

Utilizing the T3H38/v-M8 RNA switch system, we achieved in vivo regulation of AAV-mediated luciferase expression over a period of 43 weeks and precise control of AAV-mediated expression of erythropoietin to physiological level in mice (Zhong et al. Nature Biotechnology. 2020). This study provides a proof of concept that RNA switches could expand the use of AAV gene therapy to some major common diseases. On the basis of this study, our lab has been developing and optimizing modular and generalizable RNA switch systems and utilizing our optimized switch system to develop tunable AAV-hormone gene therapies. More details will be shared here as soon as we can.

RNA switch-regulated AAV-ACE2-decoy as a long-lasting vaccine-like anti-coronavirus prophylaxis for high-risk individuals

The continuous emergence of SARS-CoV-2 variants with increased immune evasion capabilities presents a great challenge on developing a long-lasting COVID-19 prophylaxis. ACE2-Ig, a recombinant fusion protein of human ACE2 ectodomain with antibody Fc, can function as an ACE2 decoy to neutralize diverse SARS-CoV-2 variants, as well as a wide range of other coronaviruses that also utilize ACE2 as an essential receptor. AAV-delivered tunable ACE2-Ig gene therapy that utilize transduced somatic cells as a bio-factory to conditionally produce and secrete ACE2-Ig into bloodstream is therefore a promising pathway to a long-lasting vaccine-like prophylaxis against diverse ACE2-utilizing coronaviruses of pandemic potential, for high-risk (e.g. the elderly and immunocompromised) individuals. Since the outbreak of the COVID-19 pandemic, our lab has been studying cross-species receptor usage of multiple coronaviruses and have developed two potent ACE2-Ig proteins, ACE2-Ig-95 and ACE2-Ig-105/106 (Lu et al. bioRxiv. 2023; under review). These two proteins showed potent and robust neutralization activities against diverse SARS-CoV-2 variants including Omicron, with an average IC₅₀ of up to 37 pM, in pseudovirus neutralization assays. In a stringent lethal SARS-CoV-2 infection mouse model, therapeutic use of either of the ACE2-Ig proteins markedly lowered lung viral load by up to ~1000 fold, prevented the emergence of clinical signs in >75% animals, and increased animal survival rate from 0% (untreated) to >87.5% (treated). ACE2-Ig-95 and ACE2-Ig-105/106 are good lead payload molecules for a tunable AAV-based anti-coronavirus prophylaxis.