Excision BioTherapeutics to Present Positive Data from its HSV-1 Keratitis Program, EBT-104, at the ASGCT 2024 Annual Meeting

• Oral presentation to discuss CRISPR-associated gene editing inactivating herpes virus
• Two poster presentations highlight the potential of EBT-104 for the treatment of HSV-1 Keratitis, demonstrating near-complete virus elimination in vitro and a significant reduction in viral shedding in vivo
  
• Company will also present data on its next generation AAV vector technology featuring an
  all-in-one AAV-Cas vector configuration with high gene editing activity and AAV productivity
  
  

SAN FRANCISCO, April 22, 2024 (GLOBE NEWSWIRE) -- Excision BioTherapeutics, Inc. (“Excision”, the “Company”), a clinical-stage biotechnology company developing CRISPR-based therapies to cure serious latent viral infectious diseases, today announced that it will present positive data from its herpes simplex virus-1 keratitis program, EBT-104, as well as an overview of a next-generation AAV delivery vector, at the American Society of Gene & Cell Therapy (ASGCT) 2024 Annual Meeting taking place May 7-11, 2024 in Baltimore, Maryland.

Herpes Simplex Keratitis (HSK), caused by the infection of herpes simplex virus type 1 (HSV-1) in the cornea, is a major cause of blindness worldwide. Although current anti-HSV-1 therapies interfere with viral DNA replication, they do not eliminate HSV-1 reservoirs or prevent recurrence. CRISPR/Cas-mediated gene editing can potentially address the underlying causes of the disease by directly eliminating the latent HSV-1 reservoirs.

Excision’s EBT-104 is a CRISPR-based gene therapy that is being developed as a potential cure for HSK. EBT-104 utilizes a CRISPR/Cas gene editing system to inactivate the latent HSV-1 virus.

“This program is part of our mission to develop curative, CRISPR-based therapies for infectious diseases of high unmet medical need by combining our novel gene editing approach, proprietary virology platform, and our expertise in genetic engineering,” said Daniel Dornbusch, Chief Executive Officer of Excision. “The multiple presentations reflect the significant progress we have made in leveraging our comprehensive platform to identify CRISPR/Cas guide RNA pairs which effectively eliminate the HSV-1 genome DNA and viral titer in vitro and reduce viral shedding in vivo. We look forward to presenting these positive data from our EBT-104 program at this year’s ASGCT Annual Meeting.”

Mr. Dornbusch continued, “In addition, we will showcase next-generation all-in-one CRISPR/Cas vector configurations with high gene editing activity, high yield, purity, and vector genome integrity. These newly engineered vectors can be manufactured at scale, demonstrating a significant increase in manufacturing yield and activity.”

The details of the presentations are below:

Title: CRISPR/Cas9-mediated gene editing for herpes simplex virus reduces viral reactivation in a latent rabbit keratitis model
Session Type: Poster presentation
Session Title: Ophthalmic and Auditory Diseases
Abstract: 1633
Presenter: Nadia Amrani, Excision BioTherapeutics
Location: Exhibit Hall
Date/Time: May 10, 2024, 5:30 to 7:00 pm (EST)

Recurrent reactivation of HSV-1 and the infection of the cornea in HSK cause corneal opacity, edema, corneal scarring, and neovascularization, which can lead to irreversible vision impairment and blindness. Currently, there is no vaccine available for HSV-1, and while nucleoside analog treatments inhibit virus replication, they cannot eliminate the infection. By targeting two critical genes in HSV-1, ICP0, and ICP27, and using the dual cut method, we induced large deletions in the viral genome preventing viral escape post-DNA repair. In Vero cells with all-in-one AAV2 vectors expressing SaCas9 and paired gRNAs nearly eliminated the viral load and titer. In vivo, using corneal scarification in a latent rabbit model of HSV-1 keratitis, our approach led to a remarkable reduction of over 60% in viral shedding from the treated rabbit eyes. Intravenous (IV) administration of AAV9 vector expressing SaCas9 and paired gRNAs in rabbits showed undetectable viral shedding in all but one rabbit following HSV-1 reactivation. Even at a low AAV dose (6E+12 VG/kg) following IV delivery, we observed substantial levels of AAV vector genomes in the trigeminal ganglia (TG), where the latent HSV-1 is located. Additionally, there was a notable decrease in HSV-1 viral DNA and LAT RNA in the TG of treated rabbits compared to the control group, indicating effective targeting of the latent HSV-1 reservoir.

Title: CRISPR/CasX-Mediated Gene Editing as a Therapeutic Approach for HSV-1 Keratitis
Session Type: Poster presentation
Session Title: Gene Disruption and Excision
Abstract: 1188
Presenter: Guoxiang Ruan, Excision BioTherapeutics
Location: Exhibit Hall
Date/Time: May 09, 2024, 5:30 to 7:00 pm (EST)

Utilizing a high-throughput lentiviral screening method, we identified effective guide RNA pairs capable of disrupting the HSV-1 viral genome and inactivating the HSV-1 virus. We created a LentiCasX2-2xgRNA library comprised of the 440 CasX2 gRNA pairs formed from the gRNAs targeting the specific genes. Lentiviral vectors derived from this library were introduced into an HSV-1 gain-of-signal knock-in reporter HEK 293FT cell line containing the ICP27 and ICP0 consensus sequences. Successful excision of ICP27-ICP0 was facilitated by blasticidin drug selection followed by FACS harvesting. The region containing the paired CasX2 gRNAs was then PCR amplified and sequenced using Nanopore long-range sequencing. The top two CasX2 gRNA pairs from the screen were tested in Vero cells infected with the HSV-1 virus. To slow down the quick replication of the HSV-1 virus, the Vero cells were treated with valacyclovir for 24 hours post-infection followed by additional four days of culturing to allow the HSV-1 replication. Compared to the empty vector control, the two CasX2 gRNA pairs reduced HSV-1 genome DNA and viral titer by over 96%.

Title: Develop Next Generation All-in-One AAV Vectors for CRISPR/Cas Gene Editing with Paired Guide RNAs
Excision Program: Next Generation Vectors
Session Type: Poster presentation
Session Title: AAV Vectors – Virology and Vectorology
Abstract: 966
Presenter: Wenwen Huo, Excision BioTherapeutics
Location: Exhibit Hall
Date/Time: May 09, 2024, 5:30 to 7:00 pm (EST)

This study demonstrates the development of an all-in-one AAV-Cas vector configuration with high gene editing activity and AAV productivity. Fourteen dual-guide, all-in-one AAV-SaCas9 configurations were designed by modifying the composite, location, and orientation of the SaCas9 expression cassette and the expression cassettes of the two guide RNAs that target the viral long terminal repeats (LTR) and the Gag gene of HIV-1. A human beta-actin gene-derived stuffer sequence in the backbone of the AAV plasmids to minimize AAV backbone was introduced and AAV crude lysates were prepared to transduce an HIV-1 knock-in reporter cell line and examine the HIV-1 excision efficiency induced by the paired HIV-1 guide RNAs. The seven most efficient AAV-SaCas9 plasmids were further co-transfected in Hela cells along with the HIV-1 plasmid pNL4-3. Among these, two configurations exhibited the highest SaCas9 and guide RNA expression levels resulting in superior HIV-1 excision efficiency. Bioreactors test on one top AAV9-SaCas9 configuration (with an AAV vector size of 4.7 kb) and two top AAV9-PlmCasX configurations (both with an AAV vector size of 4.5 bp) demonstrated a 3-10 fold increase in AAV yield and infectious titer compared to the first generation AAV construct with over 90% PacBio sequencing reads mapped to the vector genomes and AAV plasmid backbone packaging below 1%. These findings underscore the potential of the dual-guide, optimized all-in-one AAV vectors as a versatile delivery platform for CRISPR/Cas gene editing therapeutics.

Title: Selecting highly conserved and specific guide RNAs for CRISPR/CasX-mediated gene editing of the HSV-1 genome
Session Type: Oral presentation
Session Title: On- and Off-target Method Development
Abstract: 256
Presenter: Meltem Isik, Excision BioTherapeutics
Location: Ballroom 3
Date/Time: May 10, 2024, 2:00 to 2:15 pm (EST)

The large size (~152kb) of the HSV-1 genome and its genetic diversity (1–4% variation in nucleotide identity between viral genomes) pose significant challenges for genomics understanding. Ongoing research uncovers new genotypes, underscoring the importance of considering HSV-1 genetic diversity in selecting lead guides for therapeutic genome editing. In this study, we used the CRISPR/CasX gene editing system to inactivate the latent HSV-1 virus by co-targeting two essential HSV-1 genes, ICP0 and ICP27. To perform guide design, we built a Herpersviridae family CDS database to select highly conserved guide RNAs. 22 PlmCasX guide RNAs targeting ICP0 and 20 PlmCasX guide RNAs targeting ICP27 were found with a perfect sequence match in over 70% of HSV-1 strains. They were also found with perfect match to the consensus sequences of ICP0 and ICP27. The 440 guide RNA pairs formed with these 42 PlmCasX guide RNAs were tested in tissue culture for DNA excision efficiency. The most efficacious guide RNA pairs were evaluated for possible off-target editing using homology-dependent and homology-independent methods. The results were analyzed using in-house developed bioinformatics pipelines and targeted amplicon sequencing. The off-target assessment identified several highly specific PlmCasX guide RNAs that showed no detectable off-target editing activity.

About Excision BioTherapeutics, Inc.
Excision BioTherapeutics, Inc. develops CRISPR-based medicines as potential cures for serious viral latent infectious diseases. The Company’s proprietary, multiplexed gene editing platform unites CRISPR technologies with a novel gene editing approach which demonstrated the ability to stop viral replication. Excision’s pipeline targets large, underserved markets including herpes simplex virus (HSV-1 keratitis), hepatitis B virus (HBV), and human immunodeficiency virus-1 (HIV-1). Excision’s foundational technologies were developed in the laboratories of Dr. Kamel Khalili at Temple University and Dr. Jennifer Doudna at the University of California, Berkeley. For more information, please visit www.excision.bio.

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