Optimized nonviral gene delivery for primary urinary renal progenitor cells to enhance cell migration

Abstract

Progressive loss of glomerular podocytes during kidney disease leads to irreversible kidney failure, and is exacerbated by the fact that podocytes are terminally differentiated epithelial cells and unable to proliferate. Regeneration of lost podocytes must therefore derive from nonpodocyte sources. Human urine‐derived renal progenitor cells (uRPCs) are attractive podocyte progenitors for cell therapy applications due to their availability from patient urine and ability to migrate to injured glomeruli and differentiate into de novo podocytes after intravenous administration. Because gene delivery has emerged as an important strategy to augment the functionality and survival of cell therapies prior to injection, in this work we optimized nonviral gene delivery conditions (cell density, DNA dose, % FBS, and transfection material composition) to primary uRPCs. Using the cationic polymer‐peptide conjugate VIPER for gene delivery and the Sleeping Beauty transposon/transposase constructs for gene integration, we optimized transfection parameters to achieve efficient transgene expression (up to 55% transfected cells) and stable transgene expression (>65% integration efficiency) lasting up to 10 days. With these methods, we transfected uRPCs to overexpress CXCR4, an important chemokine receptor that mediates uRPC migration to the kidneys after intravenous injection, and demonstrate that CXCR4‐uRPCs exhibit enhanced migration compared to mock‐transfected cells.

Urine-Derived Epithelial Cells as Models for Genetic Kidney Diseases

“…These trials are particularly useful in podocyte loss syndromes, since glomerular podocytes are terminally differentiated epithelial cells and unable to proliferate. Human urine-derived renal progenitor cells are a good potential source of podocyte progenitors for cell therapy applications, as they can differentiate in vivo at sites of injured glomeruli into de novo podocytes after intravenous administration [10,11,217].…”

Materiomically Designed Polymeric Vehicles for Nucleic Acids: Quo Vadis?

“…238 Pun and co-workers optimized transfection parameters for polymer−peptide conjugates to induce progenitor cell migration to injured kidney sites. 239 Given the vast design space within polymer synthesis as well as polyplex assembly (buffer composition, pH, addition, sequence, N/P ratio, nucleic acid concentrations), it is surprising that DoE approaches are not widely used to streamline formulation development. Despite its routine application in the biopharmaceutical industry, lack of familiarity with statistical tools and reluctance to abandon traditional optimization approaches such as "one-factor-at-atime" have impeded the adoption of DoE in academic research.…”

“…Dahlman and co-workers departed from their pooled screening approach to implement a DoE-inspired cluster-based iterative screening approach to map relationships between lipid nanoparticle chemical descriptors and lung delivery to develop nebulized mRNA formulations . Pun and co-workers optimized transfection parameters for polymer–peptide conjugates to induce progenitor cell migration to injured kidney sites . Given the vast design space within polymer synthesis as well as polyplex assembly (buffer composition, pH, addition, sequence, N/P ratio, nucleic acid concentrations), it is surprising that DoE approaches are not widely used to streamline formulation development.…”