Gene Therapy 201

Scientist in laboratory looking through a microscope

Viral gene therapy vectors may be broadly divided into two categories: integrating and non-integrating. Integrating viruses stably incorporate their genetic cargo into the chromosomal DNA of the host cells, whereas non-integrating viruses persist outside of the chromosomal DNA. A benefit of integrating viruses is that the modified DNA is passed to daughter cells, providing durable gene expression over the life of that cell and that of its progeny. However, this comes at the risk of heritable disruption or activation of endogenous gene function. Non-integrating viruses, conversely, do not modify the DNA of the host cells, and instead persist as extrachromosomal episomes capable of gene expression but not replication. As a result, these episomes are diluted out during cell division, providing gene expression for the life of the parent cell, but decreasing expression in each successive generation of progeny.

Odylia Therapeutics utilizes adeno-associated virus (AAV), which is a non-integrating virus. This is particularly well suited for gene modification of the retina, which is composed of largely non-dividing tissue, alleviating the constraints posed by episome dilution during cellular propagation. To create a therapeutic AAV particle, the genome of wildtype AAV is stripped of all coding genes and replaced by recombinant DNA encoding for both regulatory control regions and cDNA of the therapeutic gene. The only remaining viral DNA is a structure known as an inverted terminal repeat (ITR) which is necessary for both particle formation and intracellular DNA reassembly after cellular infection. Since these recombinant particles are devoid of all viral coding regions, they are incapable of replicating after infecting the host cell.

Recombinant AAV is produced in host cells by introducing DNA containing the ITR-flanked gene expression cassette in trans with separate DNA particles containing the necessary viral structural and assembly proteins. The viral capsid is first formed, and then, using viral assembly proteins, the ITR-flanked expression cassette is loaded into the viral particle. The fully formed viral particles are then released from the cell and isolated using standard bioprocessing techniques. These purified viral particles are then delivered to the patient, either directly to the target tissue, such as the retina, or systemically though peripheral injection. Upon reaching the target tissue, the virus binds to the cell surface and is trafficked to the nucleus of the cell. There, the DNA is uncoated from the capsid and converted into stable episomal structures. These DNA structures are then expressed using the provided cis regulatory elements, allowing the therapeutic protein to be produced by the patients own cells.

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