Gene modified CD34+ cells with increased ALDH1 expression confers in vitro protection against cyclophosphamide

Authors:

Ramesh Halder
Andrew Davis
Tung Nguyen
Spriha Singh
Loosineh Avakians
Joseph Cohen
Zeus Ochoa
David Hardy
Mark Dybul
Serhat Gümrükcü

Gene modified CD34⁺ cells with increased ALDH1 expression confers in vitro protection against cyclophosphamide Ramesh Halder¹, Andrew Davis¹, Tung Nguyen¹, Spriha Singh¹, Loosineh Avakians¹, Joseph Cohen¹, Zeus Ochoa¹, David Hardy³, Mark Dybul¹, and Serhat Gümrükcü²

¹ Enochian BioSciences Inc., Los Angeles, CA, ² Seraph Research Institute, Los Angeles, CA, ³ Johns Hopkins University School of Medicine Baltimore, MD 

INTRODUCTION

?#8226;烢ngraftment remains a critical challenge for hematopoietic stem cell (HSC) transplant. 

?#8226;烥enetic modification of HSC to improve engraftment with non-myeloablative cytotoxic treatment regimens could substantially improve engraftment.

?#8226;烝ldehyde dehydrogenase-1 (ALDH1) is known to confer enhanced cellular resistance to cytotoxic agents, including cyclophosphamide (CY).

煙•Certain diseases, e.g. HIV, have known genetic modifications that can be curative.

?#8226;烼herefore, infusion of HSC with genetic modifications known to be curative and to increase expression of ALDH1 combined with low-dose CY could significantly increase engraftment and treatment success.

MECHANISM OF ACTION

• Without the ALDH1 enzyme, cells exposed to mafosfamide (MFA), a metabolite of CY that does not require liver enzymes, undergo apoptosis.
• In cells with basal levels of ALDH1 expression, a dose dependent number of cells will survive MFA treatment.
• In cells transduced to overexpress ALDH1, more cells will survive MFA treatment at any dose when compared with wild type cells.

METHODS AND MATERIALS

•CD34+TF1a cell line (ATCC, CRL-2451) and primary huCD34+ cells were transduced with lentiviral vectors overexpressing ALDH1 (LV800), or ALDH1/shRNA-CCR5/C44 (LV801) (Figure 2). shRNA-CCR5 causes a knock-down of CCR5, a key HIV co-receptor, and C44 expresses an HIV fusion inhibitor C-peptide.

•Transduction was evaluated using qPCR and vector copy number (VCN) analysis. AldeFluor kits (StemCell) were used to assess ALDH1 enzymatic activity.  Transduced cells were treated with 0.0 – 50 μM of (MFA) and cultured for 48, 72, 96, and 168 hours.

•Results  were evaluated for each condition using the ChemoMetec NC-200 cell counter, or CountBright Absolute Counting Beads added before flow cytometry evaluation. Cells were stained using CD34 Ab, and propidium iodide or Annexin-V with 7-AAD and analyzed for viability using the MACSQuant-10 flow cytometer.

•For flow cytometry evaluation we employed hierarchical gating for all lymphocytes, then all singlets; of all singlets we recorded data for number of viable cells. Absolute cell counts were calculated using the formula:

RESULTS

?#8226;烶rimary huCD34+ cells displayed extensively similar growth curves in culture when untransduced, and when transduced with LV800, or LV801 (Figure 3).

?#8226;烼F-1a cells transduced with LV800 or LV801 demonstrated an increase in cells with high levels of ALDH1 activity of 25-35 fold, and 5-10 fold, respectively, as compared with untransduced cells (Figure 4, A).

?#8226;烼ransduced TF-1a cells treated with 3.12 μM of MFA showed higher total live cell counts of 2-3 fold at 48 hours, 1.5 fold at 72 hours, and 5 fold at 96 hours (Figure 4, B).

?#8226;烶rimary huCD34+ cells transduced with LV800 or LV801 and treated with 1.25 μM MFA showed higher absolute cell counts by 50,000 cells and 20,000 cells, respectively, 7 days after treatment.

?#8226;烪igh dose MFA (10 or 20 μM) overcame chemoprotective effect of the overexpression of ALDH1 (Figure 5).

DISCUSSION

煙•Transduction with lentiviral vectors that induce ALDH1 overexpression successfully protects both TF-1a and primary huCD34+ cells from cytotoxicity with low dose MFA.

?#8226;烼herefore, over-expression of ALDH1 may increase the engraftment of gene modified HSCs in a non-myeloablative HSCs transplant.

?#8226;烼he effects of MFA were observed with vectors (ENOB-HV-1 800 and 801) containing cassettes both to increase ALDH1 expression and to protect them from HIV expression. Therefore, the approach could be a potential approach to treat or cure HIV.

?#8226;烼he results should be verified with in vivo systems, with the goal of increasing engraftment levels for infused modified CD34+ cells by using administration of cyclophosphamide in humanized mice.

CONCLUSION

?#8226;Overexpression of ALDH1 provided chemoprotection to CD34+ cell line and human stem/progenitor cells when exposed to low dose mafosfamide, a metabolite of cyclophosphamide.

?#8226;烥enetically modifying autologous human stem/progenitor cells to overexpress ALDH1 along with other genetic modifications known to treat or cure diseases, for example HIV, could lead to increased engraftment with the use of low dose cyclophosphamide.

For more information about the in vivo demonstration of these in vitro results see Poster #431