CRISPR-enhanced adipocyte browning to improve glucose tolerance in obesity and diabetes

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The long term goal of this project is to advance a CRISPR-based technology that genetically modifies human white adipocytes to a “brown-like”, thermogenic phenotype with greatly enhanced ability to promote metabolic health when implanted into obese, diabetic mice. Gene targeting is accomplished in this project without viral or expression vectors, but rather by delivering purified, endotoxin-free Cas9 protein and sgRNA complexes to adipocyte progenitors with nearly 100 percent efficiency. The central hypothesis we will test is that simultaneous CRISPR-based disruption of two distinct “browning suppression” pathways will synergize and convert abundant human white adipocytes to large numbers of rare, fully “brown- like”, therapeutic adipocytes. An attractive transcriptional suppressor target is NRIP1/RIP140, which when deleted by CRISPR in adipocytes causes 100-fold increases in expression of UCP1 and other “BAT” genes. We recently found Nrip1KO mouse or human adipocytes improve metabolic health when implanted in obese mice. An attractive suppressor target in a second thermogenic pathway (cAMP signaling) is the inhibitory RIIb subunit (gene Prkar2) of protein kinase A, which when deleted also upregulates UCP1. Remarkably, we found agonist stimulation of cAMP in Nrip1KO adipocytes upregulates UCP1 and beneficial factors in synergistic mode, approaching a full BAT phenotype. Specific Aim 1 will test the central hypothesis in vitro, leveraging lessons we learned during development of this CRISPR method under pilot Department of Defense funding. First, not all sgRNAs that cause indels in the target gene Nrip1 actually delete the protein. Thus, screening 10 to 20 Prkar2 sgRNAs will be performed in mouse and human adipocytes to define the most efficient at RIIb protein deletion and upregulation of UCP1, with least off-target effects. Second, conditions in which multiple sgRNAs function at near 100 percent efficiency will be used, allowing analysis of adipocytes with single Nrip1KO or Prkar2KO vs adipocytes with double Nrip1/prkar2KO. Efficacy of “brown-like” phenotype induction will be analyzed by cAMP determinations, Western blot and RT-PCR for thermogenic and beneficial secreted proteins, RNAseq signatures and glycolytic and oxygen consumption rates. We will thus determine whether deletion of RIIb plus NRIP1 proteins in mouse and human adipocytes is synergistic, converting white adipocytes to a near BAT phenotype. In Specific Aim 2, these CRISPR-engineered mouse and human adipocytes will be implanted in HFD fed wild type mice or HFD fed immune-compromised NSG mice, respectively. These studies will determine whether double Nrip1KO/Prkar2KO adipocytes are most effective in forming brown-like, implanted adipose depots, including extent of innervation and vascularization (light sheet microscopy). The implanted mice will be evaluated for glucose tolerance and energy expenditure (hyperinsulinemic clamp and metabolic cages). This project will thus define whether CRISPR-engineered mouse and human adipocytes doubly depleted of RIIb plus NRIP1 proteins have greater therapeutic potential than single KO or unmodified adipocytes.