Introduction

The past decade has witnessed the discovery and rapid advancement of novel genetic engineering technologies. While genome editing is not a new concept, directing precise sequence changes at desired sites has remained a challenge.

In recent years, new tools based on a bacterial CRISPR-associated protein-9 nuclease (“CRISPR/Cas9”) have generated considerable excitement. CRISPR and CRISPR-associated genes are essential for adaptive immunity in select bacteria and archaea, enabling the organisms to defend against invading viruses.

CRISPR/Cas9 was initially discovered in the 1980s in E. coli, but its function wasn’t confirmed until 2007. Recent research has demonstrated the CRISPR/Cas9 system’s potential to be efficient, adaptable, and highly flexible, making it an ideal gene editing tool.

Agenovir was founded based on technology developed in the laboratory of Stephen Quake, Ph.D.., a professor of Bioengineering and Applied Physics at Stanford University. Agenovir is using state-of-the-art nucleases, including CRISPR/Cas9, to target viral DNA for disruption. By disrupting intracellular viral DNA, it may be possible to treat and eliminate persistent viral reservoirs.

Guide RNA
Target Viral DNA
Cas9 Protein

CRISPR/Cas9 Complex

Targeted DNA Disruption

Agenovir’s unique CRISPR/Cas9 strategy does not require donor DNA or host repair. The targeted disruption of viral genes is anticipated to be sufficient to kill an infected cell.

Agenovir’s CRISPR/Cas9 complex includes:
  • The Cas9 protein, which acts like a pair of molecular scissors to knockout viral DNA
  • Guide RNA that directs the Cas9 protein to a specific viral DNA sequence

Potential Advantages

Agenovir seeks to combine the benefits of prior nuclease technologies, such as zinc finger nucleases (“ZFNs”) and transcription activator-like effector nucleases (“TALENs”), to create a platform that offers a variety of advantages including:

  • High specificity and potency
  • Potential curative treatment
  • Potential utility in both in vivo and ex vivo applications
  • Ability to scale and optimize at speed
  • Ability to target multiple DNA sites simultaneously
ZFNs TALENs CRISPR CRISPRi Agenovir*
Nuclease Target Characteristic GC rich Leading T PAM motif PAM motif TBD
Ease of Generating Target Sequence Empirical,

no clear rule

Easy Easy Easy Easy
Target Site Length 9 nt pair 16 nt pair 10-20 nt 10-20 nt 16-18 nt
Cloning Payload 2 x 2 kb 2 x 4 kb 1 x 5.5 kb 1 x 5.5 kb <5 kb
Ease of Cloning Moderate Difficult Easy Easy Easy
Off Target Common Rare Moderate Moderate; but safer TBD

* Desired properties of proprietary nuclease to be developed by Agenovir

Proof-of-Concept

Agenovir has generated in vitro data for several viruses, including Human papillomavirus (“HPV”) and Epstein-Barr virus (“EBV”), that demonstrate infection-specific cell death following delivery of nucleases to human cells.

HPV 16-Specific CRISPR/Cas9 Kills
HPV-16+ Cancer Cells
CRISPR/Cas9 Kills EBV+ Cancer Cells
While Sparing EBV- Cancer Cells

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Targeted viral genome editing