Repair Drive: AAV Therapy with Selective Expansion of Transgenic Cells
An AAV therapy approach that dramatically improves the number of cells in the liver harboring a permanent copy of the therapeutic transgene
Monogenic liver diseases negatively impact the quality of life for many patients worldwide, necessitating costly treatments and liver transplants, which carry tremendous risk and are not always curative. Adeno-Associated Viral (AAV) vector technology is a powerful gene therapy approach with three current FDA-approved therapies. Translating AAV gene therapy to the liver has been challenging due to the episomal (non-integrating) nature of recombinant AAV vectors, as well as adaptive immune responses to the hepatocytes (liver cells) receiving the virus. Both of these factors limit the duration of clinical benefit, given that hepatocytes have an average lifetime of only 200-300 days under normal circumstances. While some success has been achieved in the short term, particularly for diseases such as Hemophilia A and B, achieving sustained and life-long correction remains a critical unmet need.
Targeted, permanent insertion of genes into “safe harbor” genetic loci present a solution to this problem. CRISPR/Cas9 technology can be used in conjunction with AAV delivery methods, to precisely insert therapeutic genes into the genome of the corrected cells. Targeted transgene insertion with AAV is a particularly attractive gene therapy approach, as a single transgene can be used to treat many patients, irrespective of specific underlying mutations. Integration into the genome allows for the use of endogenous promoters, saving valuable packaging space in the AAV vector. Perhaps most importantly, cells with the integrated transgene will divide and pass it on to daughter cells, ensuring permanent expression of the therapeutic protein. Capitalizing on this approach requires a method to drive the gene-corrected hepatocytes to expand and regenerate the liver.
To expand the corrected hepatocytes in an efficient and selective manner, the inventors use short interfering (siRNA) to inhibit an essential metabolic gene to condition the liver. They then provide the antidote within a gene cassette that includes a modified version of the essential gene as well as the therapeutic gene. CRISPR/Cas9 technology can be used to increase the frequency of transgene insertion through homology directed repair. Following gene editing, siRNA is delivered on a monthly basis to stress the liver cells. This allows the cells which have integrated the antidote cassette to divide and repopulate the liver tissue. In essence, this approach dramatically improves the number of cells in the liver harboring a permanent copy of the therapeutic transgene. A high protein diet can be used to increase the selective pressure on the liver and accelerate expansion of the corrected hepatocytes. Conversely, a clinically approved drug can be used to re-route the metabolic pathway as a safety switch. Once selection is complete, the patient will express the therapeutic protein of interest throughout their liver, without the introduction of foreign selectable markers or loss of normal metabolic activities. In essence, the liver is reprogrammed to that of a person without the genetic disease, using a patient’s own cells.
Together, this system constitutes “Repair Drive” a novel method to selectively integrate a therapeutic gene into liver cells using established AAV delivery methods, amplify the corrected cells in the host tissue, and control the cells’ growth using clinically approved methods.
Stage of Development
Repair Drive has been tested using an in vivo mouse model where the antidote cassette containing the essential gene and therapeutic gene were delivered by AAV for genetic insertion into a “safe harbor” locus by CRISPR/Cas9 technology. Inhibition of the essential gene through small interfering RNA promoted robust expansion of the corrected hepatocytes.
De Giorgi M, Li A, Hurley A, Barzi M, Doerfler AM, Cherayil NA, Smith HE, Brown JD, Lin CY, Bissig KD, Bao G, Lagor WR. Targeting the Apoa1 locus for liver-directed gene therapy. Mol Ther Methods Clin Dev. 2021 Apr 24;21:656-669. https://doi.org/10.1016/j.omtm.2018.11.009
Pankowicz F.P., Barzi M., Kim K.H., Legras X., Martins C.S., Wooton-Kee C.R., Lagor W.R., Marini J.C., Elsea S.H., Bissig-Choisat B., Moore D.D., Bissig K.D. Rapid Disruption of Genes Specifically in Livers of Mice Using Multiplex CRISPR/Cas9 Editing. Gastroenterology. (2018) Aug 28. Pii. S0016-5085(18)34924-2. PMID: 30170115; DOI: https://doi.org/10.1053/j.gastro.2018.08.037
- Uses clinically tested current state-of-the art AAV delivery methods.
- Gene editing method validated with in vivo mouse models.
- Utilizes novel CRISPR/Cas9 technology for permanent integration of antidote cassette, as opposed to temporary AAV episome presence in cells.
- System is flexible and compatible with other delivery methods (i.e., lipid nanoparticles for CRISPR/Cas9, siRNA, antisense oligonucleotides).
- Targeted transgene insertion is generalizable to many patients with mutations in the same gene.
- A unique safe harbor locus uses a strong promoter to drive expression of the therapeutic protein from native regulatory elements.
- Introduces new concept of disrupting an endogenous metabolic gene to promote expansion of corrected cells within host tissue.
- Avoids the need for non-human proteins in selection of hepatocytes
- Uses clinically approved drugs and methods to control corrected cell growth.
- Normal liver physiology is restored after selection.
- Ensures AAV delivered transgenes are present in a larger fraction of the liver and permanently expressed for the life of the patient.
Available for exclusive license
- Patent application submitted