Minimal conditioning in Fanconi anemia promotes multi-lineage marrow engraftment at 10-fold lower cell doses.

BACKGROUND: Gene therapy approaches for the treatment of Fanconi anemia (FA) hold promise for patients without a suitably matched donor for an allogeneic bone marrow transplant. However, significant limitations include the collection of sufficient stem cell numbers from patients, the fragility of these cells during ex vivo manipulation, and clinically meaningful engraftment following transplantation. With these challenges in mind, we were interested in determining (i) whether gene-corrected cells at progressively lower numbers can successfully engraft in FA; (ii) whether low-dose conditioning facilitates this engraftment; and (iii) whether these cells can be selected for post-transplant. METHODS: Utilizing a well characterized mouse model of FA, we infused donor bone marrow from healthy heterozygote littermates that are unaffected carriers of the FANCA mutation to mimic a gene-corrected product, after administering low-dose conditioning. Once baseline engraftment was observed, we administered a second, very-low selective dose to determine whether gene-corrected cells could be selected for in vivo. RESULTS: We demonstrate that upfront low-dose conditioning greatly increases successful engraftment of hematopoietic corrected cells in a pre-clinical animal model of FA. Additionally, without conditioning, cells can still engraft and demonstrate a selective advantage in vivo over time following transplantation, and these corrected cells can be directly selected for in vivo after engraftment. CONCLUSIONS: Minimal conditioning prior to bone marrow transplant in Fanconi anemia promotes the multi-lineage engraftment of 10-fold fewer cells compared to nonconditioned controls. These data provide important insights into the potential of minimally toxic conditioning protocols for FA gene therapy applications.

Development of Methane-Utilizing Mixed Cultures for the Production of Polyhydroxyalkanoates (PHAs) from Anaerobic Digester Sludge.

The fundamental components required for scaling up the production of biogas-based biopolymers can be provided through a single process, that is, anaerobic digestion (AD). In this research, the possibility of enriching methane-utilizing mixed cultures from the AD process was explored as well as their capability to accumulate polyhydroxyalkanoates (PHAs). For almost 70 days of operation in a fed-batch cyclic mode, the specific growth rate was 0.078 ± 0.005 h-1 and the biomass yield was 0.7 ± 0.08 mg-VSS/mg-CH4. Adjusting the nitrogen levels in AD centrate resulted in results comparable to those obtained with a synthetic medium. The enriched culture could accumulate up to 51 ± 2% PHB. On the other hand, when the culturing medium was supplemented with valeric acid, the enriched bacteria were able to produce polyhydroxybutyrate- co-valerate (PHBV) up to 52 ± 6% with an HV percentage of 33 ± 5%. Increasing the valeric acid concentration in the culturing medium above 100 mg/L decreased the overall amount of PHBV by 60%, whereas the number of HV units incorporated was not affected. Changing the methane-to-oxygen ratio (M/O) from 1:1 to 4:1 caused an almost 80% decline in PHB accumulation. In addition, M/O had a significant effect on the fraction composition of PHBV at different valeric acid concentrations.