Focus on CRISPR Delivery Methods

December 27, 2023 by David Yoder

The next frontier for cell and gene therapy

Getting a sense of the direction of therapeutic development is an important element in seeing opportunities and overcoming obstacles that are either in place or are anticipated. The most telling message for the direction of CRISPR-based cell and gene therapy coming from CRISPR 2.0 in Boston this year is one that flowed through all the speaker presentations, delivery of a gene editing modality in vivo.

The delivery theme began with the pre-conference workshop where attendees heard talks from Dr. Ross Wilson, Adjunct Professor at UC Berkley; Dr. Gregory Newby, Asst. Professor at Johns Hopkins; Dr. Qiyu Wang, Director at Vesigen Therapeutics; Dr. Rammohan Devulapally, Director, Nonviral Delivery at Life Edit Therapeutics; and Dr. Jing Liao, Director, Development and Operations at Alexion Pharmaceuticals.

Exploring new delivery paths
As translational medicine research moves toward in vivo modalities, a significant effort is being placed on delivering therapies targeting specific organs, tissues, and cells, while avoiding nontargets. The delivery system should be scalable, cargo agnostic, transient, low toxicity, endosomal escape, and redoseable or nonimmunogenic.

Additional goal for some delivery systems is evasion of neutralizing antibodies (Ab) in the case of AAV, or diffusion and access across the blood brain barrier (BBB) in other instances. Multiple delivery methodologies are being developed to meet these needs, including peptide-mediated delivery, engineered Virus-Like Particles (eVLPs), Extracellular Vesicles (EVs), Tissue-Specific Lipid Nanoparticles (LNPs), and Engineered Adeno-associated Virus (AAV) vectors.

Dr. Wilson, session chair and former Doudna Lab member, presented Peptide-Mediated Efficient Delivery of CRISPR Enzymes based on the Ross Lab’s system of peptide-enabled RNP* Delivery for CRISPR engineering or PERC. This delivery method uses amphiphilic delivery peptides for non-toxic delivery of RNPs to edit knock outs (KO), knock-ins (KI), and adenine base editor (ABE) modalities. The benefits of the PERC system are that it is transient, scalable, nontoxic, small, and cargo agnostic.

The second presentation, Efficient & Precise In Vivo CRISPR Delivery With Engineered Virus-Like Particles, was given by Dr. Newby, a former member of the Liu Lab at Harvard. In this talk, we learned how the lab developed a system, eVLP, that is able to express and package an RNP, including modified Cas enzymes like a base editor, into a deliverable capsule with similar infiltration to a lenti virus without the risk of DNA integration. The current version is specific to the liver but could potentially be used in more isolated environments like the retina or evolved further to be tissue specific.

We also enjoyed an introduction to EVs by Dr. Wang as he talked about ARMMs (ARRestin-domain 1 Mediated Microvesicles) technology in his talk, Extracellular Vesicles: An Emerging Promising Delivery Vehicle. In this delivery system, scientists take advantage of naturally occurring EVs to reduce the possibility of immunological responses by up regulating ARRestin Domain Containing Protein 1 (ARRDC1) in a cell line to increase loading capacity of tagged molecules into the EV. This system is scalable with high potency, potentially tunable to specific cells, and redoseable, targeting many of the needs for in vivo gene editing delivery.

Dr. Devulapally presented Enabling Tissue-Specific In Vivo Targeting With Lipid Nanoparticles. As a scientific community, we have been introduced to LNPs through recent manufacturing strategies to deliver an mRNA-based vaccine. Here, the LNP is used to deliver the components for gene editing as either RNPs or mRNA. Dr. Devulapally’s team has discovered LNPs that can specifically target the lung, spleen, or liver, based on the pKa of the lipids. In this system, a pKa above 9 targets the lung, where a pKa below 6 targets the spleen, and a pka of 6-7 targets the liver. Additionally, naturally occurring lipid associating molecules can be used to take advantage of uptake through cholesterol or apolipoprotein E (ApoE).

The final presentation was Next-Generation Engineering of AAV Vectors, by Dr. Liao. Here we learned of efforts to overcome several inherent limitations of AAV, which include an inability to redose, immunity from prior exposure, and neutralizing Abs (NAbs). The team used an enhanced DNA shuffling screen using 12 natural serotypes to develop a chimeric AAV that is not recognized by Abs in either humanized mice or non-human primates. This system, designated sL65, was used to identify 12 variants with mutations in the VR1 domain, important for tropism and Ab recognition, that resulted in a NAb evading capsid. This technology opens the door for potential redosing that current AAV capsids would not support.

Each of these technologies brings CRISPR-based cell and gene therapies a step closer to in vivo therapies, which is critical for addressing many genetic disease indications that can only be treated in terminally differentiated, nondividing cells. Further, these technologies are developed to allow for specific edits to target cells, without impacting or editing untargeted cells and tissues. It is amazing to see how far CRISPR technology has come from a mere decade ago when it was first realized how useful CRISPR can be for site-directed editing in mammalian cells, the future of cell and gene therapy is looking very promising.

• Visit our CRISPR solutions web page
• View our webinar, CRISPR Genome Editing Solutions from Discovery to Clinic
• View our webinar, CRISPR Cures 2033
• Read Jacqueline Rodriguez’s CRISPR 2.0 recap blog post
• Want to know more about this topic? Contact Dave Yoder
• Have an idea for a topic? Let us know!

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