Collaborating Investigator: Hans-Peter Kiem, MD, PhD 
Affiliation: Fred Hutchinson Cancer Research Center  

Funding Source: NIH
Grant Number: U54DK106829 
Project Period: 08/01/15 – 07/31/26

Significance

The goal of CP #5 is to replace viral gene therapy biotechnology tools with non-viral gene therapy biotechnology tools and Dr. Kiem is developing cell therapeutics for patients. Specifically, the focus is on hematopoietic stem cell (HSC) gene therapy as a promising treatment option for a variety of genetic diseases affecting the hematopoietic system, from HIV/AIDS, to cancer, to rare genetic diseases. Ex vivo HSC gene therapy is inefficient, expensive, and limited in availability and viral gene therapy has cargo size limitations and manufacturing concerns. To overcome these limitations, next generation non-viral gene therapy will be conducted using NIMs that can be surface-modified to enhance on-target specificity (as in Aim 3 of TR&D2). To accomplish the goals, gene editing cargos will be delivered to HSCs in vivo using CD90-targeted NIMs to achieve the targeting. Both full-length antibodies (Ab) and Ab-derived single chain variable fragments will be evaluated for targeting, and if successful in a humanized mouse model, the NIMs will be further evaluated in a NHP model as the next future step.

Approach

Aim 1: Develop CD90-targeted NIMs for gene delivery.  NIMs can be decorated with full-length antibodies (Ab) or Ab-derived single chain variable fragments (scFvs). The Kim lab has used an anti-CD90 Ab-producing hybridoma cell line to produce full-length Abs. The Kim lab has further sequenced the antigen-binding region and engineered anti-CD90 scFvs in close collaboration with the Strong Lab at the Fred Hutch. We will chemically modify CD90-targeted NIMs using anti-CD90 Abs/derivatives as well as engineered anti-CD90 scFvs to deliver GFP mRNA and base editors into HSCs, characterize physicochemical properties of CD90-targeted NIMs, and evaluate their delivery to cells.

Aim 2: Optimize NIM-mediated in vivo HSC gene editing in the humanized mouse model. Systemic administration of gene therapy agents can lead to off-target effects. We will evaluate the biodistribution and on-target delivery of surface engineered NIMs from the first aim by performing injections directly into the BM stem cell compartment and blood in a humanized NBSGW mouse model. Luciferase, GFP, and base editor loaded NIMs will be used to track the biodistribution and target-specificity of NIMs. Next, surface-modified NIMs will be evaluated for enhanced targeting of CD90+ HSCs and impact on biodistribution. Finally, base editor-loaded NIMs will be used to knock out a therapeutic gene. Mice will be monitored longitudinally and comprehensively analyzed at necropsy for on-/off-target editing. If successful, future work would progress to a NHP model.

Push-Pull relationship:

Push: The NCBIB is synthesizing libraries of innovative biodegradable nanoparticles useful for intracellular delivery broadly, including as tools and to facilitate gene therapy/gene editing. Further, TR&D2 is also focused on surface engineering to enable targeted delivery to blood cells. This CP helps to push the technology out to enable cellular therapies for additional immune and non-immune applications.

Pull: Dr. Kiem’s lab has seen first-hand the limitations of viruses for ex vivo gene therapy and gene editing and seeks new technologies to facilitate non-viral gene therapy and gene editing for improved safety, efficacy, and manufacturing. We will work together on surface conjugated biodegradable nanoparticles for genetic manipulation of cells in vivo in animal models. The Kiem lab will provide feedback and expertise on the use of TR&D2 materials for new targeting applications and use in additional animal models.