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Introduction:
Red blood cells can theoretically be used as a platform for therapeutic proteins because
they are both long lived and naturally occur in circulation. The inherent advantages of
erythrocytes lend themselves to mimicking the cell surface of the helper T-lymphocyte,
which is the natural target of the HIV-1 virus. Modified blood cells could theoretically act
as a decoy target for HIV. As a decoy, these cells would be capable of binding to and
internalizing the retrovirus, effectively neutralizing viruses it contacts while in the blood
stream. Through the addition of the CD4, CXCR4 and CCR5 proteins, erythrocytes will
share many characteristic surface markers of the helper T-lymphocyte. Only the cell
surface marker CD4 has been added in previous research to test this therapeutic
strategy. Advances in understanding may allow the addition of the CXCR4 and CCR5
proteins, to more effectively mimic the membrane of the T-lymphocyte. A possible
method for the insertion of these proteins is gene therapy using a retrovirus that targets
the CD34+ stem cells that give rise to red blood cells. The proposed research develops a
test of new methods of stem cell transformation can yield erythrocytes that more closely
resemble helper T–lymphocytes and are subsequently more effective in neutralizing
various strains of HIV-1
•Will a lentiviral vector be able to insert full length copies of CD4, CXCR4
and CCR5 genes into CD34+ stem cells?
•Will the addition of CD4, CXCR4 or CCR5 cells surface markers interfere
with differentiation into erythrocytes?
•Will the addition of the proteins CXCR4 and CCR5 significantly increase
the red blood cells ability to absorb different varieties of HIV-1?
•Full length insertion of CD4 into erythrocytes was first
accomplished via electroinsertion, which will fuse with HIV-1 in
vitro (1) (Figure 3.)
•The chemokine receptors CCR5 and CXCR4 were discovered to
mediate entry of HIV-1 into CD4+ cells after research into CD4RBC’s was abandoned (2,3)
•It was found that CD4-negative cells (like erythrocytes)
efficiently bind HIV-1 via cell surface heparins, and transfer these
viruses to T-cells. (4)
•CXCR4 and CCR5 using strains of HIV-1 are found to exhibit
differential pathogenesis in vivo (5).
•Efficient lentiviral vectors for gene therapy were developed and
targeted to the CD34+ stem cell progenitors of erythrocytes (6,7).
Experimental Design:
Hematopoiesis:
(1) Purify CD34+cells from
Mobilized Peripheral Blood.
Expected Results:
(2) Insert treatments via a
lentiviral vector.
Treatments:
(3) Differentiate by placing
on semisolid matrix of
methylcellulose.
(4) Supplement with IL-3
SCF, EPO, and GM-SCF.
(5) Grow and separate
mature erythrocytes.
Confirm Treatments with
Flow Cytometry.
Figure 1. Depiction of the HIV-1 lifecycle
showing the importance of CXCR4 and
CCR5 to initial binding and fusion. (Image
taken from www.thebody/nami/cycle.html)
Figure 2. Overview of hematopoiesis: In order to
Introduce specific cell markers to the human
erythrocyte, lentiviruses will be employed in order to
Insert the genetic information into stem cells.
This must be done because mature erythrocytes
lack nuclei, and are therefore incapable of
producing the Protein.
(6) Inject treatments with
isolated X4 and R5 strains
of HIV-1 that have been
fluorescently labeled.
Fig 4. A. A typical wild type retrovirus: The
components for the virus can be removed
from the virus in a producer cell line to
produce the retroviral vector shown in B.
which can then act as a vector for the gene
of interest without further viral replication.
(Source www.gmu.edu/.../ 385-Ch10bGeneTherapy/img019.jpg)
Figure 3. Results from pervious research
indicating HIV-1 is absorbed into CD4
baring erythrocytes (1).
Flouresence Dequenching of Treated
Erythrocytes
1)Control, erythrocytes
derived in vivo, no
additions
2) Control, erythrocytes
derived in vitro, no
additions
(7)Place samples under a
spectroflorometer to assay the amount of
florescence dequenching. This will
indicate how much HIV has been
absorbed by each cell.
(3) RBC-CD4
(4) RBC-CD4+CXCR4
(6) RBCCD4+CXCR4+CCR5
4) Add CD4 and CXCR4 Via
lentivirus
6) Add CD4, CXCR4 and
CCR5 via lentivirus
(2) Blood derived in
vitro
(5) RBC-CD4+CCR5
3) Add CD4 Via lentivirus
5) Add CD4 and CCR5 Via
lentivirus
HIV-1 (mixed)
HIV-1 (R5)
HIV-1 (X4)
(1)Normal blood
Treatments
Primary Research questions:
Review of literature:
0
10
20
30
40
50
DQ%
Figure 5. Expected results: Each treatment group is tested for
florescence dequenching represented in this figure. The treated
erythrocytes should show both increased uptake of the virus as well
as specificity for viruses that bind the markers inserted into them.
Literature Cited:
(1)
Ziera, Micheal. 1991 “Full-length Cd4 electroinserted in the erythrocyte membrane: as a long lived inhibitor of infection by human immunodeficiency virus.” Proc Natl
Acad Sci U. S. A. May 15:88: (see p4409-4413)
(2)
Deng, H. 1996 “Identification of a major co-receptor for primary isolates of HIV-1” Nature. 1996 June 20: 381 (6584): 661-6.
(3)
Dragic, T. “HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5.” Nature 312: 667-673.
(4)
Gene, G. “CD4-Negative Cells Bind Human Immunodeficiency Virus Type 1 and Efficiently Transfer Virus to T Cells.” Journal of Virology, September 2000, p. 8550-8557,
vol. 74, NO. 18
(5)
Berkowitz, Robert D. “CCR5 and CXCR4- Utilizing Strains of Human Immunodeficiency Virus Type 1 Exhibit Differential Tropism and Pathogenesis In Vivo,” Journal of
Virology, December 1998, p. 10108-10117, Vol 72, No. 12
(6)
Geronimi, Fabian. “Highly Efficient Lentiviral Gene Transfer in CD34+ and Cd34+/38-/lin- Cells from Mobilized Peripheral Blood after Cytokine Prestimulation.
(7) Grande, Alexis. “Transcriptional Targeting of Retroviral Vectors to the Transuded Hematopoetic Stem Cells” Blood, Vol (3 No.10 (May 15), 1999: pp3276-3285.