Recombinant Adeno Associated Virus Based Gene Delivery: A Technique for Transgene Delivery and Cardiac-Specific Expression in Rodent Model

Recombinant adeno-associated virus, rAAV, is a small, single-stranded DNA virus genetically engineered to target a specific cell type and transport its genetic cargo to the host cell nucleus for gene expression.

Begin by restraining a cryo-anesthetized mouse pup. Sterilize the back skin and subcutaneously inject the rAAV vector carrying the desired transgene along a cardiac-specific promoter. The vector gets absorbed into the vascular system and reaches distant organs, including the heart.

Here, the recombinant virus attaches to the surface of the cardiomyocyte and gets internalized via endocytosis into an endosome. Within the endosome, the viral capsid partially disassembles, exposing the viral lytic proteins. This protein induces pores in the endosomal membrane, through which the vector escapes into the cytoplasm.

The vector travels across cytoplasmic microtubules and enters through the nuclear pore. Once in the nucleus, the viral capsid disassembles to release its genome. The single-stranded DNA carrying the target gene is then converted into double-stranded DNA.

Following DNA transcription, the produced mRNA moves into the cytoplasm. Here, the mRNA undergoes translation, expressing the target protein in the cardiomyocytes. Observe tissue samples from different organs under a fluorescence microscope to determine the efficiency and tissue specificity of the gene delivery to the heart.