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C6 - Gene Targeting and Gene Correction New Technologies

1210: Synthetic AAV DNA Mediates High-Efficiency Homology-Directed Genome Editing

Type: Poster Session

Poster Board Number: 1210
Presentation Details
Session Title: Thursday Posters: Gene Targeting and Gene Correction New Technologies






AAV-based genome editing offers a promising alternative to CRISPR methods due to its enhanced safety profile and precision. Unlike CRISPR, AAV vectors integrate genetic material into the host genome in a more controlled manner, reducing the risk of off-target effects and unintended mutations. However, AAV-based genome editing is very inefficient, often reported below 0.1%. To overcome this limitation in efficiency, a synthetic AAV DNA system has been developed. Synthetic AAV DNA is a single-component, completely synthetic alternative to CRISPR/Cas9 or AAV-based genome editing tools that takes advantage of many of the inherent properties and versatility of AAV-based genome editing while bypassing its limitations (low editing efficiencies and manufacturing costs). In addition, synthetic AAV DNA is not bound to the payload size limitations of a standard AAV system, allowing for targeting of previously untreatable genetic diseases. Synthetic AAV DNA is produced utilizing enzymes in a cell-free system and results in a linear closed-ended double-stranded DNA molecule with full AAV ITRs at each end (Fig1). Genome editing with the synthetic AAV DNA does not require exogenous proteins or guide RNAs for editing and does not induce double strand breaks in genomic DNA. This eliminates on-target cutting of unedited alleles and off-target cutting effects resulting in unintended mutations. Genome editing is directed to the target sequence by homology arms matching the target sequence within the synthetic AAV DNA sequence. In vitro experiments utilizing a P2A-GFP synthetic AAV targeting the highly expressed ALB locus in HepG2 cells have consistently demonstrated GFP gene addition efficiencies between 40-50%. By placing GFP in-frame with the ALB gene, GFP expression is controlled by native ALB gene regulatory elements (Fig2). Gene addition in this manner demonstrates the potential applications for treating hemophilia for Factor VIII or Factor IX deficiencies. The synthetic AAV DNA system can also be used to repair point mutations and deletions. A synthetic AAV was designed to target and swap a TA pair to an AT pair at the AAVS1 locus. This substitution removes an NheI site and allows for editing efficiency to be visualized by restriction analysis. Editing rates at the AAVS1 locus in HeLa cells are currently ~30% for this two base-pair swap. Base substitutions of this manner have potential to repair point mutations like those responsible for sickle cell disease. Junctional analysis for both the GFP gene addition and the two-base substitution, reveal seamless editing with no inclusion of AAV ITR sequences detected. The synthetic AAV DNA system can also be used as a gene therapy vector to express genes over 4.7 kb in size. An example synthetic AAV ABCA4 (6.8 kb) expression vector was assembled to show this feature of the system. An ABCA4 synthetic AAV could be used to treat Stargardt’s disease. Other oversized synthetic AAV DNA vectors have potential to treat diseases such as Usher’s (16.6 kb) and DMD (11.1 kb). A suitable delivery system will be needed to deliver the synthetic AAV system to the appropriate target tissues.



Plain Language Summary
AAV-based genome editing offers a promising alternative to CRISPR methods due to its enhanced safety profile and precision.

Christopher Luis Chavez

Visgenx, Santa Cruz, CA"

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