1. Hematopoietic stem cell based gene therapy for HIV diseases
Dong Sung An, M.D., Ph.D Associate professor UCLA School of Nursing UCLA AIDS Institute

2. Long-term goals
Advance stem cell based gene therapy research.
Develop a new therapy for cure for HIV infected individuals.

3. Limitations in the current treatment for HIV infection
No cure.
Rapid rebound of viremia if patients stop medication.
Everyday life long medication
Treatment adherence is difficult.
Side effects.
Medication costs ($100,000-$400,000 for one patient’s life).
Emergence of multi-drug resistant HIVs.
Limited treatment access world wide.
No vaccine

4. A Man is Cured of AIDS
Photo Credit:

5. HIV entry into CD4 T lymphocytes is
mediated by the chemokine CCR5 receptor

6. Natural HIV resistance by CCR532/32 mutation
CCR5 32/32 homozygous mutation
1% in Caucasian population
No CCR5 expression
Naturally protected from HIV-1 infection

7. Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation
Hutter et.al. N Engl J Med Feb 12;360(7):692-8.
CCR5-32/ 32
BM Donor (HIV-)
Acute Myeloid Leukemia
Patient (HIV+)
Bone Marrow
transplant
This “Berlin Patient”
was cured from HIV.
A clinical case report just published last month in New England Journal of Medicine for a long term control of HIV by CCR5 delta 32 stem cell transplant provided an evidence that inhibition of CCR5 expression has a great potential to treat HIV infected patients. A 40 years old HIV infected individual developed an acute myeloid leukemia in Germany. A doctor intentionally chose a bone marrow donor with CCR5 delta 32 homozygous mutation to treat leukemia and HIV in the patient. After the bone marrow transplant, nearly 100% of human hematopoietic cells were replaced with the CCR5 negative donor cells.
HARRT was discontinued after the bone marrow transplant. HIV RNA and DNA became undetectable at 68 days post transplant and remained negative for 20months.
This result is very encoulegding for us. But, how we can identify delta 32 bone marrow donor? It is rate. The doctor identified one from whole bone marrow donor in Germany. It could be more difficult to find a donor in other ethnicity since CCR5 delta 32 mutation is primarily in Caucasians and almost none in other ethnicities.
Nearly 100% replacement with the CCR5 negative donor cells.
HAART was discontinued after BM transplant.
HIV RNA and DNA became undetectable at 68 days post-transplant
and remained negative for 5 years.

8. A novel HIV cure therapy Develop a hematopoietic stem cell based gene therapy for long term control or HIV cure by a single treatment

9. Develop anti-HIV gene therapy strategies using autologous hematopoietic stem cells

10. RNA interference (RNAi)
siRNA (20nt)
CCR5 mRNA
AAAAn
I decided to use RNA interference technology to reduce CCR5 expression. used small interferening to reduce CCR5 expression in human cells. can be induced by small interfering RNA in 20nt length can induce sequence specific messenger RNA degradation in cells. interference
I designed several siRNAs that are complemenary to human CCR5 messenger RNA. siRNA needs to be stably expressed in cells. RNA is fragile siRNA.
RNAi
Induce sequence specific
mRNA degradation
Remove CCR5 from cell surface
Inhibit HIV infection

11. Stable CCR5 knock down by RNAi
to confer resistance to R5 tropic HIV-1 infection
CCR5
Lentiviral vector
CCR5
mRNA
shRNA
Qin XF, An DS, Chen ISY, D Baltimore, PNAS, 2003

12. Modeling RNAi-mediated CCR5 knockdown in NOD/SCID humanized BLT mouse
CCR5 shRNA (EGFP)
Irradiation
NOD/SCID
Hu BLT mouse
Human CD34+
Stem cells
No shRNA (mCherry)

13. Efficient CCR5 down regulation in EGFP+ CD4+ T cells in the humanized BLT mouse model
Thy/Liv
Bone Marrow
Spleen
Lymph Node
Lung
Small Intestine LPL
CCR5
EGFP
CCR5 expression was efficiently down regulated in EGFP marked CD4+ T cells in all tissue analyzed, but not in GFP negative cells or in mCherry marked cells, showing the specificity.
mCherry
Shimizu. S et. al. Blood 2010

14. Selective advantage of EGFP+ CCR5 knock down CD4+ T cells in peripheral blood
CCR5 shRNA
no shRNA
CCR5 tropic HIV-1
HIV-1 NFNSX SL9 (R5)
HIV-1 NL4-3 (X4)
After infection with the R5 tropic HIV-1, the CD4/CD8 ratio remains higher in EGFP+ cells relative to mCherry+ cells.
In contrast, during X4 tropic HIV-1 infection, The CD4/CD8 ratio was dropped. Therefore CCR5 down regulation by shRNA prevents depletion of CD4+ T cells during R5 tropic HIV-1 infection in vivo in peripheral blood.
Unpublished

16. Mr. Timothy Brown “Berlin Patient” The man was cured of HIV
I will
I promise to develop
a cure therapy for
HIV patients!