Screening chimeric GAA variants in preclinical study results in hematopoietic stem cell gene therapy candidate vectors for Pompe disease.

Pompe disease is a rare genetic neuromuscular disorder caused by acid α-glucosidase (GAA) deficiency resulting in lysosomal glycogen accumulation and progressive myopathy. Enzyme replacement therapy, the current standard of care, penetrates poorly into the skeletal muscles and the peripheral and central nervous system (CNS), risks recombinant enzyme immunogenicity, and requires high doses and frequent infusions. Lentiviral vector-mediated hematopoietic stem and progenitor cell (HSPC) gene therapy was investigated in a Pompe mouse model using a clinically relevant promoter driving nine engineered GAA coding sequences incorporating distinct peptide tags and codon optimizations. Vectors solely including glycosylation-independent lysosomal targeting tags enhanced secretion and improved reduction of glycogen, myofiber, and CNS vacuolation in key tissues, although GAA enzyme activity and protein was consistently lower compared with native GAA. Genetically modified microglial cells in brains were detected at low levels but provided robust phenotypic correction. Furthermore, an amino acid substitution introduced in the tag reduced insulin receptor-mediated signaling with no evidence of an effect on blood glucose levels in Pompe mice. This study demonstrated the therapeutic potential of lentiviral HSPC gene therapy exploiting optimized GAA tagged coding sequences to reverse Pompe disease pathology in a preclinical mouse model, providing promising vector candidates for further investigation.

Rapid and efficient in vitro generation of pancreatic islet progenitor cells from nonendocrine epithelial cells in the adult human pancreas.

The absence of efficient and directed methods for the differentiation of adult pancreatic progenitor cell populations to pancreatic islet cells has raised doubts concerning the regeneration potential inherent in the adult pancreas. Relatively low levels of islet cell differentiation have been reported using adult pancreatic cells in vivo and in vitro. In the present study, we initially enriched for a nonendocrine epithelial component of the adult human pancreas and defined conditions that are permissive to islet cell differentiation in vitro. Sequential progression of cell differentiation in the permissive conditions allowed for incremental evaluation of changes occurring in the cell population. Optimization of the differentiation process, for the efficient production of islet endocrine cells, was accomplished by identifying specific factors and culture conditions that increased islet progenitor production 250-fold. Ultimately, 85% percent of the nonendocrine epithelial cells isolated from human pancreatic tissue and cultured in the optimized conditions for 8 days, readily re-expressed pancreatic duodenal homeobox-1 (Pdx1). Sixty-five percent of these Pdx1-expressing cells were capable of additional islet endocrine cell differentiation. This represents a significant advancement in the differentiation of an adult pancreatic progenitor cell population in vitro and suggests that the nonendocrine compartment of the human pancreas remains an important cell resource for the generation of transplantable islets to treat diabetes.

Scalable passaging of adherent human pluripotent stem cells.

Current laboratory methods used to passage adherent human pluripotent stem cells (hPSCs) are labor intensive, result in reduced cell viability and are incompatible with larger scale production necessary for many clinical applications. To meet the current demand for hPSCs, we have developed a new non-enzymatic passaging method using sodium citrate. Sodium citrate, formulated as a hypertonic solution, gently and efficiently detaches adherent cultures of hPSCs as small multicellular aggregates with minimal manual intervention. These multicellular aggregates are easily and reproducibly recovered in calcium-containing medium, retain a high post-detachment cell viability of 97%±1% and readily attach to fresh substrates. Together, this significantly reduces the time required to expand hPSCs as high quality adherent cultures. Cells subcultured for 25 passages using this novel sodium citrate passaging solution exhibit characteristic hPSC morphology, high levels (>80%) of pluripotency markers OCT4, SSEA-4, TRA-1-60 andTRA-1-81, a normal G-banded karyotype and the ability to differentiate into cells representing all three germ layers, both in vivo and in vitro.

Human amnion-derived multipotent progenitor cell treatment alleviates traumatic brain injury-induced axonal degeneration.

To identify a viable cell source with potential neuroprotective effects, we studied amnion-derived multipotent progenitor (AMP) cells in a rat model of penetrating ballistic-like brain injury (PBBI). AMP cells were labeled with fluorescent dye PKH26 and injected in rats immediately following right hemispheric PBBI or sham PBBI surgery by ipsilateral i.c.v. administration. At 2 weeks post-injury, severe necrosis developed along the PBBI tract and axonal degeneration was prominent along the corpus callosum (cc) and in the ipsilateral thalamus. Injected AMP cells first entered the subventricular zone (SVZ) in both sham and PBBI rats. Further AMP cell migration along the cc only occurred in PBBI animals. No significant difference in injury volume was observed across all treatment groups. In contrast, treatment with AMP cells significantly attenuated axonal degeneration in both the thalamus and the cc. Interestingly, PKH26-labeled AMP cells were detected only in the SVZ and the cc (in parallel with the axonal degeneration), but not in the thalamus. None of the labeled AMP cells appeared to express neural differentiation, as evidenced by the lack of double labeling with nestin, S-100, GFAP, and MAP-2 immunostaining. In conclusion, AMP cell migration was specifically induced by PBBI and requires SVZ homing, yet the neuroprotective effect of intracerebral ventrical treatment using AMP cells was not limited to the area where the cells were present. This suggests that the attenuation of the secondary brain injury following PBBI was likely to be mediated by mechanisms other than cell replacement, possibly through delivery or sustained secretion of neurotrophic factors.