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