The desirability of CpG depletion of AAV vectors for the purpose of reducing the immune response to the vector and establishing long-term transgene expression is a hot topic of interest to many gene therapists. The presentation will discuss the mechanism by which immunostimulatory CpG motifs limit transgene expression (presenting data from both preclinical and clinical studies) as well as strategies for the future design of AAV vectors.

Directed evolution, coupled with in vivo selection in primates, enables the systematic exploration of the AAV capsid fitness landscape, identifying gene therapy vectors with novel tissue tropism, preexisting neutralizing antibody resistance, and/or minimal liver tropism to reduce the risk of systemic toxicity. Here, we describe the selection and preclinical characterization of vectors that form the basis of gene therapeutics for ophthalmology, cardiology and lung disease indications.

Duchenne muscular dystrophy is one of the most common muscular dystrophies caused by mutated forms of the dystrophin gene. Novel approximations are trying to treat these patients with gene therapy using viral vectors. We have coated AAV particles, increasing the transduction efficiency in the several muscle cell lines tested. Moreover, we show a decrease in the interaction with neutralizing antibodies opening the way to a repeated administration for systemic treatment.

We identified an artificial miRNA scaffold derived from mouse miR-33 that shows equally effective gene silencing, reduces off-target activity by ten-fold, and produces fewer truncated genomes in comparison to conventional shRNA. In addition, the miR-33 scaffold can be expressed from inducible or tissue- or cell-type specific promoters. The miR-33 scaffold provides a valuable tool to effect on-target, potent silencing delivered via rAAV and other viral vectors for research and clinical applications. To facilitate the applications of miR-33 scaffold, we developed a user-friendly tool that enables investigators to easily design target cassettes that consider new miR-33 scaffold design rules.

As the list of potential applications for AAV-mediated gene therapy grows, so does demand for capsids tailored to address new indications. Here, we comprehensively evaluate the tropisms of 118 naturally occurring and engineered AAV capsids as a pooled library by multiple routes of administration into both male and female mice, as well as cynomolgus macaques. As such, we identified unique patterns of transduction based on tissue type, species, and sex.

Viral vectors for gene delivery are highly effective and commonly used, but they are also burdened by safety concerns, high costs, and inhibitory immune responses. Non-viral vehicles like cationic lipids and polymers are attractive alternatives, but typically less efficient. This seminar will discuss the use of mRNA-sequencing to identify host cell genes that may be responsible for inhibiting the delivery and/or expression of non-viral transgenes.

Therapeutic CRISPR genome editing is currently limited by toxicity and efficiency. We have developed high-efficiency large gene insertions targeted by Cas9 in primary human immune cells. We found that anionic polymers stabilize Cas9 RNPs into nanoparticles; adding truncated Cas9 target sequence (tCTS) at the ends of the HDRT further enhances nuclear import. Together these dramatically improve editing efficiency even at reduced HDRT doses yielding marked increases in viable edited cells.

The use of viral vectors for stable gene delivery is costly and time consuming. With the uptick in CAR-T and CAR-NK cell therapies there is an increasing need for non-viral solutions. We utilize TcBuster, a HAT family transposase. However, the initial performance of TcBuster in immune cells was undesirable. Through direct evolution, we engineered a hyperactive enzyme TcBuster that allows us to achieve favorable integration rates while using less plasmid DNA.

Lipid-based nanoparticles (LNPs) have emerged as a leading delivery system for nucleic acid therapies and have been used for clinically relevant delivery of RNA-based drugs. While these advances are exciting, systemic delivery of LNP therapies to non-hepatocytes in vivo remains a challenge. This is in part due to the way LNPs are discovered. To address this need, we developed technologies to simultaneously quantify how thousands of LNPs deliver in vivo.

The potential of systemic AAV9 gene delivery to treat neurological diseases has been demonstrated by transformative results in SMA patients and rapid approval of Zolgensma. However, large doses are required, and this success will be challenging to replicate in other neurological diseases as neuronal transduction in brain is modest. We have developed a transient BBB disruption that increases AAV9-mediated gene expression in CNS by two to three orders of magnitude.