In vivodelivery is a major barrier to the use of molecular tools for gene modification. Here we demonstrate site-specific gene editing of human cells in vivo in hematopoietic stem cell-engrafted NOD-scid IL2rγnull mice, using biodegradable nanoparticles loaded with triplex-forming peptide nucleic acids (PNAs) and single-stranded donor DNA molecules. In vitro screening showed greater efficacy of nanoparticles containing PNAs/DNAs together over PNA-alone or DNA-alone. Intravenous injection of particles containing PNAs/DNAs produced modification of the human CCR5 gene in hematolymphoid cells in the mice, with modification confirmed at the genomic DNA, mRNA, and functional levels. Deep sequencing revealed in vivo modification of the CCR5 gene at frequencies of 0.43% in hematopoietic cells in the spleen, and 0.05% in the bone marrow: off-target modification in the partially homologous CCR2 gene was two orders of magnitude lower. We also induced specific modification in the β-globin gene using nanoparticles carrying β-globin-targeted PNAs/DNAs, demonstrating this method’s versatility. In vivo testing in an EGFP- β-globin reporter mouse showed greater activity of nanoparticles containing PNAs/DNAs together over DNA only. Direct in vivo gene modification, such as we demonstrate here, would allow for gene therapy in systemic diseases or in cells that cannot be manipulated ex vivo.

Our data suggest that using an iodixanol-controlled density gradient improves the islet recovery rate in human islet isolation. On the basis of these data, we now use this purification method for clinical islet transplantation.

Pancreatic islet transplantation is a promising treatment for diabetes but still faces several challenges. Poor islet isolation efficiency and poor long-term insulin independence are currently two major issues, although donor shortage and the need for immunosuppressants also need to be addressed. We established the Kyoto islet isolation method (KIIM), which has enabled us to isolate and transplant islets even from non-heart-beating donors. KIIM involves 1) cooling the donor pancreas in situ, 2) preserving the ducts with modified Kyoto solution, 3) using a modified two-layer pancreas preservation method, and 4) adjusting the density of the density gradient centrifugation and using an iodixanol-based solution for purification. KIIM has enabled us to transplant 17 islet preparations out of 21 isolations (an 81% success rate). All transplanted islets functioned, and all transplanted patients had improved glycemic control without hypoglycemic unawareness. Recently, we used KIIM for islet isolation from a brain-dead donor at Baylor, which resulted in a very high islet yield (789,984 IE) with high viability (100% by fluorescein diacetate/propidium iodide staining and a stimulation index of 4.7). This preliminary evidence suggests that KIIM may also be promising for islet isolation from brain-dead donors. In addition, to assess engrafted islet mass, we developed a secretory unit of islet transplant objects (SUITO) index: fasting C-peptide (ng/dL) / [fasting blood glucose (mg/dL) - 63] x 1500. This simple index has enabled us to monitor the engrafted islet mass. This index should be useful when deciding whether to perform additional islet transplantations to maintain insulin independence. Poor islet isolation efficacy and poor long-term results could be resolved with ongoing research.

Inconsistent islet isolation is one of the issues of clinical islet transplantation. In the current study, we applied ductal injection to improve the consistency of islet isolation. Seven islet isolations were performed with the ductal injection of ET-Kyoto solution (DI group) and eight islet isolations were performed without the ductal injection (standard group) using brain-dead donor pancreata. Isolated islets were evaluated based on the Edmonton protocol for transplantation. The DI group had significantly higher islet yields (588,566 ± 64,319 vs. 354,836 ± 89,649 IE, p < 0.01) and viability (97.3 ± 1.2% vs. 92.6 ± 1.2%, p < 0.02) compared with the standard group. All seven isolated islet preparations in the DI group (100%), versus only three out of eight isolated islet preparations (38%) in the standard group met transplantation criteria. The islets from the DI group were transplanted into three type 1 diabetic patients and all three patients became insulin independent. Ductal injection significantly improved quantity and quality of isolated islets and resulted in high success rate of clinical islet transplantation. This simple modification will reduce the risk of failure of clinical islet isolation.