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TRANSCRIPT
Gene modified CD34+ cells with increased ALDH1 expression confers in vitro protection against cyclophosphamide
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Ramesh Halder, Andrew Davis, Tung Nguyen, Spriha Singh, Loosineh Avakians, Joseph Cohen, Zeus Ochoa, David Hardy, Mark Dybul, and Serhat Gümrükcü
Disclosure
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The following experiments were carried out by employees of Enochian Biosciences
Introduction
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� Engraftment remains a critical challenge for hematopoietic stem cell (HSC) transplant.
� Genetic modification of HSC to improve engraftment with non-myeloablative cytotoxic treatment
regimens could substantially improve engraftment.
� Aldehyde dehydrogenase-1 (ALDH1) is known to confer enhanced cellular resistance to cytotoxic
agents, including cyclophosphamide (CY).
� Certain diseases, e.g. HIV, have known genetic modifications that can be curative.
� Therefore, 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
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• 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.
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Materials and Methods
• 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) 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.
LV 800
LV 801
EF1a hALDH1-ORF
H1Pro
hCCR5shRNA EF1a hALDH1-ORF modified
C44
2.9 kB
3.5 kB
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Materials and Methods
• 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:
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RESULTS
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Primary huCD34+ cells transduced with LV800 or LV801 do not show cytotoxic effect
0 2 4 60
20
40
60
80
CD34 Cell Growth
Days PTD
Via
ble
Ce
lls
(x
10
6)
Un-Td800-Td801-Td
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ALDH1 activity and total live cell count increase in transduced CD34+TF-1a cells after MFA treatment
0.0 3.12 6.25 12.5 25 500
2
4
1020304050607080
48 Hours
MFA (µM)
% C
D34
+ Ald
eflu
or+
Un-Td800-Td801-Td
0.0 3.12 6.25 12.5 25 500
1
2
10203040506070
72 Hours
MFA (µM)
% C
D34
+ Ald
eflu
or+
Un-Td800-Td801-Td
0.0 3.12 6.25 12.5 25 500
1
2
3
10203040506070
96 Hours
MFA (µM)
% C
D34
+ Ald
eflu
or+
Un-Td800-Td801-Td
0.0 3.12 6.25 12.5 25 500
500
1000
1500
2000
2500
MFA (µM)
Tot
al L
ive
Cel
l C
oun
tUn-Td800-Td801-Td
48 Hours
0.0 3.12 6.25 12.5 25 500
400
800
1200
3000
4000
5000
MFA (µM)
Tot
al L
ive
Cel
l C
oun
t Un-Td800-Td801-Td
72 Hours
0.0 3.12 6.25 12.5 25 500
200
400
6001000
2000
3000
MFA (µM)
Tot
al L
ive
Cel
l C
oun
t Un-Td800-Td801-Td
96 Hours
A
B
• TF-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.
• Transduced 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
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Primary huCD34+ cells are protected from mafosfamide (MFA) cytotoxicity when transduced with vectors over expressing ALDH1
0 2 3 4 70
25
50
75
100
125
150
(0.0 µM)
Days after MFA treatment
Abso
lute
Cel
l Cou
nt (x
103 ) Utd
800 td801 td
0 2 3 4 70
25507580
90
100
110
120
130
(1.25 µM)
Days after MFA treatment
Abso
lute
Cel
l Cou
nt (x
103 ) Utd
800 td801 td
0 2 3 4 70
20
40
60
80
100
120
(2.5 µM)
Days after MFA treatment
Abso
lute
Cel
l Cou
nt (x
103 ) Utd
800 td801 td
0 2 3 4 70
10
20
30
40
50
60
(5.0 µM)
Days after MFA treatment
Abso
lute
Cel
l Cou
nt (x
103 )
Utd800 td801 td
0 2 3 4 70
5
10
15
20
25
304060
(10.0 µM)
Days after MFA treatment
Abso
lute
Cel
l Cou
nt (x
103 ) Utd
800 td801 td
0 2 3 4 70
2
4
6
8
10304050
(20.0 µM)
Days after MFA treatment
Abso
lute
Cel
l Cou
nt (x
103 ) Utd
800 td801 td
• Primary 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.
• High dose MFA (10 or 20 µM)
overcame chemoprotective effect of
the overexpression of ALDH1.
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Discussion:
� Transduction with lentiviral vectors that induce ALDH1 overexpression successfully protects both
TF-1a and primary huCD34+ cells from cytotoxicity with low dose MFA.
� Therefore, over-expression of ALDH1 may increase the engraftment of gene modified HSCs in a
non-myeloablative HSCs transplant.
� The 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.
� The 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.
Conclusions
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� Overexpression of ALDH1 provided chemoprotection to CD34+ cell line and
human stem/progenitor cells when exposed to low dose mafosfamide, a
metabolite of cyclophosphamide.
� Genetically 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.
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Enochian BioSciences Inc.Los Angeles, CA
• Ramesh Halder PhD
• Andrew Davis• Tung Nguyen• Spriha Singh• Loosineh Avakians• Joseph Cohen PhD• Zeus Ochoa • Mark Dybul MD
Acknowledgments Seraph Research Institute Los Angeles, CA
Serhat Gümrükcü MD PhD
Johns Hopkins University School of MedicineBaltimore, MDDavid Hardy MD
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For more information about the in vivo demonstration of these in vitro results see Poster #431
Thank you