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ABSTRACT

SummaryCurrent donor cell-dependent strategies can only produce limited “made-to-order” therapeutic natural killer (NK) cells for limited patients. To provide unlimited “off-the-shelf” NK cells that serve many recipients, we designed and demonstrated a holistic manufacturing scheme to mass-produce NK cells from induced pluripotent stem cells (iPSCs). Starting with a highly accessible human cell source, peripheral blood cells (PBCs), we derived a good manufacturing practice-compatible iPSC source, PBC-derived iPSCs (PBC-iPSCs) for this purpose. Through our original protocol that excludes CD34+ cell enrichment and spin embryoid body formation, high-purity functional and expandable NK cells were generated from PBC-iPSCs. Above all, most of these NK cells expressed no killer cell immunoglobulin-like receptors (KIRs), which renders them unrestricted by recipients' human leukocyte antigen genotypes. Hence, we have established a practical “from blood cell to stem cells and back with less (less KIRs)” strategy to generate abundant “universal” NK cells from PBC-iPSCs for a wide range of patients.

Gene delivery into the spinal cord provides a potential approach to the treatment of spinal cord traumatic injury, amyotrophic lateral sclerosis, and spinal muscular atrophy. These disorders progress over long periods of time, necessitating a stable expression of functional genes at therapeutic levels for months or years. We investigated in this study the feasibility of achieving prolonged transgene expression in the rat spinal cord through repeated intrathecal administration of plasmid DNA complexed with 25 kDa polyethylenimine (PEI) into the lumbar subarachnoid space. With a single injection, DNA/PEI complexes could provide transgene expression in the spinal cord 40-fold higher than naked plasmid DNA. The transgene expression at the initial level persisted for about 5 days, with a low-level expression being detectable for at least 8 weeks. When repeated dosing was tested, a 70% attenuation of gene expression was observed following reinjection at a 2-week interval. This attenuation was associated with apoptotic cell death and detected even using complexes containing a noncoding DNA that did not mediate any gene expression. When each component of the complexes, PEI polymer or naked DNA alone, were tested in the first dosing, no reduction was found. Using polyethylene glycol (PEG)-grafted PEI for DNA complexes, no attenuation of gene expression was detected after repeated intrathecal injections, even in those rats receiving three doses, administered 2 weeks apart. Lumbar puncture is a routine and relatively nontraumatic clinical procedure. Repeated administration of DNA complexed with PEG-grafted PEI through this less invasive route may prolong the time span of transgene expression when needed, providing a viable strategy for the gene therapy of spinal cord disorders.

Transcriptional targeting using a tissue-specific cellular promoter is proving to be a powerful means for restricting transgene expression in targeted tissues. In the context of cancer suicide gene therapy, this approach may lead to cytotoxic effects in both cancer and nontarget normal cells. Considering microRNA (miRNA) function in post-transcriptional regulation of gene expression, we have developed a viral vector platform combining cellular promoter-based transcriptional targeting with miRNA regulation for a glioma suicide gene therapy in the mouse brain. The therapy employed, in a single baculoviral vector, a glial fibrillary acidic protein (GFAP) gene promoter and the repeated target sequences of three miRNAs that are enriched in astrocytes but downregulated in glioblastoma cells to control the expression of the herpes simplex virus thymidine kinase (HSVtk) gene. This resulted in significantly improved in vivo selectivity over the use of a control vector without miRNA regulation, enabling effective elimination of human glioma xenografts while producing negligible toxic effects on normal astrocytes. Thus, incorporating miRNA regulation into a transcriptional targeting vector adds an extra layer of security to prevent off-target transgene expression and should be useful for the development of gene delivery vectors with high targeting specificity for cancer therapy.

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Gene delivery to sensory neurons of the dorsal root ganglion (DRG) offers the prospect of developing new clinical interventions against peripheral nerve diseases and disorders. Here we show that genes can be transferred to rat DRG through lumbar intrathecal injection of delivery vectors into the cerebrospinal fluid. Genes could be transferred to DRG using polyethylenimine (PEI)/DNA complexes, Lipofectamine 2000/DNA complexes, adeno-associated virus vectors, or baculovirus vectors. We also show that nerve growth factor cDNA, delivered through lumbar intrathecal injection of PEI complexes, was able to improve regeneration of transected rat sciatic nerves. These data demonstrate the viability of using an intrathecal gene delivery approach for treating peripheral neuropathies.

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