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Publications of the Week

Engineering Designer Beta Cells with a CRISPR-Cas9 Conjugation Platform

By August 28, 2020No Comments

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This week we profile a recent publication in Nature Communications from
the laboratory of Dr. Amit Choudhary (pictured) at the Broad Institute.

Can you provide a brief overview of your lab’s current research focus?

Death and/or dysfunction of insulin-secreting beta cells is a key pathological event in type 1 diabetes. We develop broadly-applicable chemical technologies with the intent of applying them to specific research avenues in beta-cell biology. Our current interest includes the development of methods for precision control of CRISPR-Cas9 based technologies. For example, we have developed various small molecules to precisely activate, inhibit, or degrade Cas9-based technologies. We apply these genome editing technologies to engineer stem-cell-derived beta cells that can withstand pathologic stresses. We also develop methods for selective delivery of molecules to beta cells. Finally, we are developing a new class of small molecules that will activate and endow new functions to the enzymes, and we will apply these molecules to understand the molecular basis of autoimmunity.

What is the significance of the findings in this publication?

Current beta cell transplantation therapies for type 1 diabetes suffer from immune rejection, resulting in loss of β-cell mass and only short-term therapeutic effects. The macroencapsulation of beta cells with a semipermeable membrane can protect them from the host’s immune system, though foreign body reaction-induced fibrosis can impair the mass transfer and viability of encapsulated cells. Anti-inflammatory cytokines, such as interleukin 10 (IL-10), can reduce fibrosis and promote long-term beta-cell survival and superior islet function. Therefore, engineered beta-cells that secrete anti-inflammatory cytokines and anti-fibrotic factors will propel the development of cell-based therapeutics for diabetes. Here, we have developed a convenient platform to conjugate molecules to Cas9 and using this platform we were able to enhance the knock-in efficiencies. Next, by hijacking the insulin expression and secretion machinery, we used our conjugation platform to efficiently engineer beta cells to secrete a non-endogenous peptide in a glucose-responsive manner, as is observed for insulin. Since the c-peptide is cleaved off during insulin processing and secreted, we hypothesized that knocking-in the desired gene into the c-peptide region of Insulin locus would enable the secretion of the inserted gene product. Using our chemically-enhanced Cas9, we efficiently engineered insulin-producing beta cells to secrete non-endogenous molecules, including IL-10, without incorporation of any viral or foreign sequences (e.g., promoter) other than that of the secreted molecule.

What are the next steps for this research?

In addition to engineering beta cells, we have successfully engineered other endocrine cells to secrete various factors. We are now optimizing conditions for efficient editing of human induced pluripotent stem cells to secrete various anti-fibrotic and anti-hypoxia factors to maximize their synergy for reduced fibrosis, and improved cell survival in in vivo models. We are also engineering these cells to become more responsive to glucose-stimulated insulin secretion.

This work was funded by:

We thank the support from Burroughs Wellcome Fund (Career Award at the Scientific Interface), DARPA (BrdiN66001-17-2-4055), and NIH (UC4DK116255, R01 DK113597, HL095722).

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