Targeting the heart: CRISPR/Cas9-VPR mediated concurrent knockout and activation to treat cardiac diseases, exemplified by a gene therapy strategy for CPVT

Thalhammer, Stefan (2024): Targeting the heart: CRISPR/Cas9-VPR mediated concurrent knockout and activation to treat cardiac diseases, exemplified by a gene therapy strategy for CPVT. Dissertation, LMU München: Faculty of Biology

Abstract

Background: Cardiovascular diseases are the leading cause of death worldwide. Lifelong restrictions and often inadequate pharmacotherapeutic treatment put a heavy burden on patients and healthcare systems and leave many patients at constant risk of fatal cardiac events. In contrast, gene therapy offers potential cures for inherited or acquired cardiac diseases with currently nonexistent or limited treatment options, including heart failure, cardiomyopathies, and channelopathies, via a single application. Recombinant adeno-associated-virus (AAV) mediated in vivo transgene delivery is currently the gold standard for gene therapy. Yet, insufficient cardiac vector delivery, the small packaging capacity of 4.7 kb, and a lack of therapeutic approaches for diseases caused by gain-of-function mutations limit clinical applications. Thus, to date, no gene therapy for cardiac diseases has been approved, which illustrates the unmet need for further development of current approaches. Aims and methods: 1) AAV capsid engineering can be used to optimize cardiac transduction efficiency and specificity. Here, the tropism of four novel AAV9-based capsid variants carrying small peptide insertions was investigated in mice. 2) By initially implementing the recently developed dual rAAV-vector approach utilizing reconstitution via mRNA trans-splicing (REVeRT) in a heart context, we aimed to enable an efficient cardiac delivery of large transgenes. 3) Moreover, CRISPR/Cas9-VPR-mediated concurrent knockdown and activation (CONNACT) by single guide RNA multiplexing was evaluated as an innovative mutation-independent strategy to treat cardiac diseases with gain-of-function mutation origin. As such, catecholaminergic polymorphic ventricular tachycardia 1 (CPVT1), which is caused by gain-of-function mutations in the RYR2 gene, was addressed via a heart-specific knockout of Ryr2 and transactivation of Ryr1 via CONNACT as a novel gene therapeutical approach. Results: 1) The cardiac transduction efficacy of the novel AAV9 variants was comparable or inferior to that of the parental AAV9 capsid when administered intraperitoneally. 2) The dual REVeRT vector technology, combined with a cardiac-specific promoter, resulted in exceptional rates of up to 76% successfully targeted cardiomyocytes. 3) In the therapeutical approach, dual REVeRT vector-mediated delivery of CIRSPR/Cas9-VPR demonstrated a proof-of-concept for CONNACT in cardiac tissue. Ryr2 knockout could be linked to ER stress and SR disruption in mature cardiomyocytes, while Ryr1 activation showed no adverse effect. However, RYR1 could not functionally compensate for RYR2. Conclusion: This study demonstrates the power of REVeRT and CONNACT as valuable technologies for the development of cardiac gene therapies but indicates that the choice of analog proteins for functional compensation requires extensive preclinical work. Furthermore, our findings shed light on the impact of RYR2 depletion and RYR1 expression in mature cardiomyocytes.

• Item Type: Theses (Dissertation, LMU Munich)
• Keywords: Gene Therapy, CPVT, Ryanodine Receptor, CRISPR/Cas9
• Subjects: 500 Natural sciences and mathematics; 500 Natural sciences and mathematics > 570 Life sciences
• Faculties: Faculty of Biology
• Language: English
• Date of oral examination: 17. July 2024
• 1. Referee: Biel, Martin
• Signature of the printed copy: 0001/UMC 32135