Identification of unique reciprocal and non reciprocal cross packaging relationships between HIV-1, HIV-2 and SIV reveals an efficient SIV/HIV-2 lentiviral vector system with highly favourable features for in vivo testing and clinical usage

Abstract

Background: Lentiviral vectors have shown immense promise as vehicles for gene delivery to non-dividing cells particularly to cells of the central nervous system (CNS). Improvements in the biosafety of viral vectors are paramount as lentiviral vectors move into human clinical trials. This study investigates the packaging relationship between gene transfer (vector) and Gag-Pol expression constructs of HIV-1, HIV-2 and SIV. Cross-packaged vectors expressing GFP were assessed for RNA packaging, viral vector titre and their ability to transduce rat primary glial cell cultures and human neural stem cells.

Results: HIV-1 Gag-Pol demonstrated the ability to cross package both HIV-2 and SIV gene transfer vectors. However both HIV-2 and SIV Gag-Pol showed a reduced ability to package HIV-1 vector RNA with no significant gene transfer to target cells. An unexpected packaging relationship was found to exist between HIV-2 and SIV with SIV Gag-Pol able to package HIV-2 vector RNA and transduce dividing SV2T cells and CNS cell cultures with an efficiency equivalent to the homologous HIV-1 vector however HIV-2 was unable to deliver SIV based vectors.

Conclusion: This new non-reciprocal cross packaging relationship between SIV and HIV-2 provides a novel way of significantly increasing bio-safety with a reduced sequence homology between the HIV-2 gene transfer vector and the SIV Gag-Pol construct thus ensuring that vector RNA packaging is unidirectional.

Figure 3

Limiting dilution RT PCR of Virion associated GFP RNA. For each viral vector, four PCR s were performed containing a target cDNA at neat, 1/10, 1/20 and 1/40 dilution. A: Lanes 1–4, HIV-1 Gag-pol + HIV-1 Vector, Lanes 5–8, HIV-1 Gag-pol + SIV vector, Lanes 9–12, HIV-1 Gag-pol + HIV-2 vector, Lanes 13–16, SIV Gag-pol + SIV vector. B: Lanes 1–4, SIV Gag-pol + HIV-2 vector, Lanes 5–8, HIV-2 Gag-pol + HIV-2 vector, Lanes 9–12, HIV-2 Gag-pol + HIV-1 vector, Lanes 13–16, HIV-2 Gag-pol + SIV vector.

Figure 4

FACS analysis of GFP expression in SV2 cells transduced with homologous and cross-packaged lentiviral vectors (10 ng of vector). Lower Right hand quadrant represents GFP positive cells. HIV-1 Gag-Pol + HIV-1GFP vector (A), HIV-1 Gag-Pol + HIV-1 cPPT-GFP vector (B), HIV-1 Gag-Pol + SIV GFP vector (C), HIV-1 Gag-Pol + HIV-2 GFP vector (D). HIV-2 Gag-Pol + HIV-2 GFP vector (E), HIV-2 Gag-Pol + SIV GFP vector (F), HIV-2 Gag-Pol + HIV-1 GFP vector (G). SIV Gag-Pol + SIV GFP vector (H), SIV Gag-Pol + HIV-2 GFP vector (I), SIV Gag-Pol + HIV-1 GFP vector (J).

Figure 6

GFP expression from HIV-2 vectors following transfection inot produced cells and in cells transduced with the packaged vectors.

Figure 7

Transduction of rat mixed glial cells with a HIV-2 based lentiviral vector packaged by SIV gag-pol. (A) GFP expression in lentivector transduced cells. (B) GFAP co-staining of astrocytes.

Figure 8

Transduction of Rat primary mixed glial cultures with Lentiviral vectors based on HIV-1 packaged by HIV-1 Gag-pol(A), HIV-1 +cPPT vector packaged by HIV-1 Gag-pol (B), HIV-2 vector packaged by SIV Gag-pol (C) and HIV-2 vector packaged by HIV-2 Gag-pol (D). Error bars indicate within experimental SEM.

Figure 9

Transduction of neural stem cells by a HIV-2 based GFP lentiviral vector packaged by SIV-2 Gag-Pol. (A) Phase contrast image through growing neurosphere (upper left). (B) Fluorescent image of neurosphere in A expressing GFP 72 hours post transduction (upper right). (C) Confocal image through neurosphere expressing GFP (lower left) (D) Neurons derived from human neurosphere 7 days post differentiation (lower right). Red represents β tubulin III, green – GFP, Hoechst stain (blue) nuclei. Arrow denotes double labelled cell. Magnification in A and B = 10×, in C = 100×, D = 40×

Figure 5

a Quantitative assessment of GFP transfer to SVC2 cells by FACS analysis using HIV-1 Gag-Pol to package gene transfer vectors based on HIV-1 (+/- cPPT sequence), HIV-2 and SIV. A range of Viral vector concentrations from 40 ng to 4 ng of Reverse Transcriptase was used. (Blank = No data). b Quantitative assessment of GFP transfer to SVC2 cells by FACS analysis using SIV Gag-Pol to package gene transfer vectors based on SIV, HIV-1 and HIV-2. A range of Viral vector concentrations from 20 ng to 4 ng of Reverse Transcriptase was used. c Quantitative assessment of GFP transfer to SVC2 cells by FACS analysis using HIV-2 Gag-Pol to package gene transfer vectors based on, HIV-2, HIV-2 and SIV. A range of Viral vector concentrations from 20 ng to 4 ng of Reverse Transcriptase was used.

Figure 1

Gag-Pol packaging constructs.

Figure 2

GFP gene transfer vectors. The dotted line indicates a deletion