A large-capacity bacterial platform for the production and targeted delivery of gene editing systems - SUMMARY
There is an unmet need for delivery systems that empower broad therapeutic use of gene editing technologies.
The power of the CRISPR/Cas system has been harnessed for a variety of gene editing approaches, and it has
been hailed as the future of therapeutic gene editing. A key impediment to the therapeutic implementation of
CRISPR/Cas-mediated gene editing strategies is the efficient delivery of the required components, i.e., two non-
coding RNAs and the large Cas9 nuclease, to target cells and tissues. Current delivery platforms, e.g., viruses
and non-viral platforms, suffer from several weaknesses, including (1) lack of targeting specificity, 2) inability to
enter cells, 3) immune activation, 4) off-target effects and limited therapeutic window, and 5) limited genetic
encoding and cargo capacity. SiVEC Biotechnologies, with support from phase I and phase II SBIR grants from
NIAID (1R43AI140243-01A1 and 2R44AI140243-02), has previously developed a bacteria-based delivery
platform to efficiently generate and deliver short-hairpin RNAs (shRNAs) to specifically targeted tissues. This
platform is not limited to shRNA delivery, and in this proposal, we will engineer it to create “SiCRISP” – a novel
platform that produces and delivers all components needed for CRISPR/Cas9-directed gene editing. We will also
perform proof-of-concept studies for its use in the delivery of gene editing machinery to clinically relevant tissues.
By applying the key components of the SiVEC delivery platform, SiCRISP overcomes several critical
shortcomings that limit the therapeutic potential of existing gene editing delivery systems: (1) it can target specific
cells and tissues, and can be administered via multiple routes, e.g., intranasal, systemic, targeted injection,
intraocular, intravaginal, among others; (2) it can enter cells and escape the endosome to efficiently deliver its
cargo; (3) it has been experimentally confirmed as non-immunogenic, and it is not recognized by host immune
cells, even after repeated delivery; (4) its mechanism does not depend on host genome integration, thus its effect
is transient, reducing off-target effects, limiting toxicity, and improving safety; (5) it has no limitation on coding
and delivery capacity (i.e., it simultaneously expresses and delivers all CRISPR/Cas9 components in one dose);
(6) it is inexpensive to produce quickly and in large quantities; (7) as an all-in-one system, it eliminates additional
manufacturing steps; (8) it can produce and deliver the components of any gene editing system (e.g., TALENs,
base editors, and meganucleases) due to its coding versatility and large coding capacity. These innovative
features of the SiCRISP delivery platform will help to realize the full potential of gene editing for the treatment of
human disease.
