STABLE INTEGRATION OF SIN TRANFER VECTORS

Abstract

The present invention relates to the field of the production of lentiviral vectors (LV) for gene therapy. More particularly, the invention relates to a system to obtain the stable integration of a Self Inactivating (SIN) lentiviral transfer vector into a packaging cell line. The SIN lentiviral transfer vector is integrated using a DNA fragment containing the Inverted Terminal Repeats (ITR) of Adeno Associated Virus (AAV), thus obtaining the stable producer cell line for SIN lentiviral vectors.

Claims

1. A system for the stable integration of a SIN lentiviral transfer vector into a packaging cell line wherein such system consists of a DNA fragment comprising, starting from the 5 end: i. The Inverted Terminal Repeat of the Adeno Associated Virus (AAV ITR) ii. A SIN lentiviral transfer vector iii. A constitutive promoter that regulates the expression of an antibiotic resistance gene iv. An antibiotic resistance gene v. The Inverted Terminal Repeat of the Adeno Associated Virus (AAV ITR)

2. A system according to claim 1 wherein the SIN lentiviral transfer vector comprises at position 5 a lentiviral LTR in which the U3 segment has been substituted by a CMV promoter and, at position 3, a deletion of the enhancer and promoter sequences of the lentiviral LTR.

3. A system according to anyone of claim 1 or 2 wherein the antibiotic resistance gene is Zeocin resistance gene

4. A system according to anyone of claims 1 to 3 wherein the promoter that regulates the expression of the resistance gene is a CMV promoter

5. A method to obtain a producer cell line for SIN lentiviral vectors comprising: i. Preparing a DNA fragment comprising, starting from the 5 end: the Inverted Terminal Repeat of the Adeno Associated Virus (AAV ITR), a SIN lentiviral transfer vector, a constitutive promoter that regulates the expression of an antibiotic resistance gene, an antibiotic resistance gene and the Inverted Terminal Repeat of the Adeno Associated Virus (AAV ITR) ii. Transfecting a packaging cell line for lentiviral vector with such DNA fragment iii. Selecting the producer cell line by culturing the cells in the presence of the antibiotic

6. A method according to claim 5 wherein the SIN lentiviral transfer vector comprises at position 5 a lentiviral LTR in which the U3 segment has been substituted by a CMV promoter and, at position 3, a deletion of the enhancer and promoter sequences of the lentiviral LTR

7. A method according to anyone of claim 5 or 6 wherein the antibiotic resistance gene employed in the system is Zeocin resistance gene

8. A method according to anyone of claims 5 to 7 wherein the promoter that regulates the expression of the antibiotic resistance gene is a CMV promoter

9. A method according to anyone of claims 5 to 8 wherein the packaging cell line is a stable packaging cell line obtained from an host cell line containing into its genome lentiviral gag/pol, lentiviral rev and an envelope protein

10. A method according to anyone of claims 5 to 9 wherein the packaging cell line further, contains lentiviral that stably integrated into the genome

11. A method according to claim 9 or 10 wherein the host cell line is a human cell line selected from HEK293, HEK293-T, HEK293-SF, TE671, HT1080 or HeLa

12. A method according to anyone of claims 9 to 11 wherein the env gene is selected from VSV-G env, MLV 4070 env, RD114 env, chimeric envelope protein RD114-TR, chimeric envelope protein RD114pro, baculovirus GP64 env or GALV env or derivatives thereof

13. A producer cell line obtainable by the method of any one of claims 5 to 12

14. A producer cell line containing into its genome at least one copy of a DNA fragment comprising of starting from the 5 end: i. The Inverted Terminal Repeat of the Adeno Associated Virus (AAV ITR) ii. A SIN lentiviral transfer vector iii. A constitutive promoter that regulates the expression of an antibiotic resistance gene iv. An antibiotic resistance gene v. The Inverted Terminal Repeat of the Adeno Associated Virus (AAV ITR)

15. A process for producing SIN lentiviral vectors comprising culturing a producer cell line containing into its genome at least one copy of a DNA fragment comprising starting from the 5 end: i. The Inverted Terminal Repeat of the Adeno Associated Virus (AAV ITR) ii. A SIN lentiviral transfer vector iii. A constitutive promoter that regulates the expression of an antibiotic resistance gene iv. An antibiotic resistance gene v. The Inverted Terminal Repeat of the Adeno Associated Virus (AAV ITR)

Description

DESCRIPTION OF THE FIGURES

[0080] FIG. 1. (a) Scheme of the SIN-GFP TV. Abbreviations: AAV-ITR, Adeno-Associated Virus-Inverted Terminal Repeat; CMV, cytomegalovirus promoter; U3, deleted U3, RRE, rev responsive element; SD, splice donor; SA, splice acceptor; , packaging signal; WPRE, Woodchuck Hepatitis Post-Transcriptional Regulatory Element; cPPT, central PolyPurine Tract; SV40-P, Simian Virus 40 promoter; zeoR, zeocin resistance gene. (b, c) Schemes of the SIN-RD114-TR LVs. Abbreviations: CMV, cytomegalovirus promoter; U3, deleted U3; IN, intron; BGI, rabbit beta-globin intron, RRE, Rev Responsive Element; A, polyA sequence; IRES, internal ribosome entry site; SD, splice donor; SA, splice acceptor; , packaging signal; WPRE, woodchuck hepatitis post-transcriptional regulatory element; cPPT, central Poly-Purine Tract.

[0081] FIG. 2. Analysis of the vector proteins in the producer cells and in the corresponding LVs. Western blot analysis of cellular extracts (35 g/sample) obtained from RD3-MolPack24 (a) and RD2-MolPack64 (b) producer cells and from the corresponding supernatants containing viral particles (100 ng p24Gag/sample). The membranes were hybridized sequentially with an anti-HIV human serum and the indicated anti-Rev, anti-Tat and anti-RD114-TR specific Abs and, after stripping, with the anti-actin Ab. The asterisk (*) indicates a non specific band.

[0082] FIG. 3. Transduction efficiency of activated peripheral blood T lymphocytes with either RD3-MolPack24 or VSV-G pseudo-typed SIN-GFP-zeo LVs. (a) Total PBMC were pre-activated with CD3/CD28 Dynabeads for 48 hours and cultured in the presence of IL-7 and IL-15. T lymphocytes were then transduced with the RD3-MolPack24 LVs at the indicated MOI and six and 14 days after transduction, GFP expression was evaluated by FACS analysis to calculate transduction efficiency. (b) Quantification of the SIN-GFP-zeo VCN in T lymphocytes of panel a) by Q-PCR using specific primer-probe sets recognizing the packaging () signal of the integrated vector and the genomic telomerase gene as control. (c) PBMC were pre-activated as in (a) and then transduced with 36 ng of p24Gag equivalents of either RD3-MolPack24 LVs or VSV-G pseudo-typed LVs carrying the SIN-GFP-zeo TV. Six and 14 days after transduction, the GFP expression was evaluated by FACS analysis. (d) Quantification of the SIN-GFP-zeo VCN in T lymphocytes of panel c) as described in (b). Results are average SEM of n=3 independent experiments. * p0.05; ** p0.01.

[0083] FIG. 4. Transduction efficiency of activated peripheral blood T lymphocyte subsets with either RD3-MolPack24 or VSV-G pseudo-typed SIN-GFP-zeo LVs. (a) Total peripheral blood mononuclear cells (PBMC) were pre-activated with CD3/CD28 Dynabeads for 48 hours and cultured in the presence of IL-7 and IL-15. T lymphocytes were then transduced with 36-ng p24Gag equivalent of RD3-MolPack24 LVs and six days after transduction, GFP expression was evaluated in CD3+/CD8+ and CD3+/CD8 T cell subsets (a), and in T cell memory subpopulations of CD3+/CD8+ and CD3+/CD8 T cell subsets (c) and (d) by FACS analysis. (b) Relative frequency of CD3+/CD8+ and CD3+/CD8 T cell subsets in LV- and mock-transduced cells. (e and f) Relative frequency of T central memory (TCM), T Stem Cell Memory (TSCM), T Effector Memory (TEM) and T Effector Memory RA+ (TEMRA) in either CD3+/CD8+ (e) or CD3+/CD8 T cell subsets (f) of LV- and mock-transduced cells. Results are mean SEM of n=4 independent experiments (day 6 p.t.). * p0.05; ** p0.01.

[0084] FIG. 5. Transduction efficiency of activated PB T lymphocytes with either RD3-MolPack24 or VSV-G pseudo-typed SIN-GFP-zeo LVs at MOI 25. (a) Total PBMC were pre-activated with CD3/CD28 Dynabeads for 48 hours and cultured in the presence of IL-7 and IL-15. T lymphocytes were then transduced with the RD3-MolPack24 LV s VSV-G pseudo-typed LVs carrying the SIN-GFP-zeo TV at MOI 25 and six and 14 days after transduction, GFP expression was evaluated by FACS analysis to calculate transduction efficiency. (b) Quantification of the SIN-GFP-zeo VCN in T lymphocytes of panel a) by Q-PCR using specific primer-probe sets recognizing the packaging () signal of the integrated vector and the genomic telomerase gene as control. Results are average SEM of n=3 independent experiments.

[0085] FIG. 6. Transduction efficiency of activated PB T lymphocyte subsets with either RD3-MolPack24 or VSV-G pseudo-typed SIN-GFP-zeo LVs at MOI 25. (a) Total PBMC were pre-activated with CD3/CD28 Dynabeads for 48 hours and cultured in the presence of IL-7 and IL-15. T lymphocytes were then transduced with the RD3-MolPack24 LVs at MOI 25 and six days after transduction, GFP expression was evaluated in CD3+/CD8+ and CD3+/CD8 T cell subsets (a), and in T cell memory subpopulations of CD3+/CD8+ and CD3+/CD8 T cell subsets (c) and (d) by FACS analysis. (b) Relative frequency of CD3+/CD8+ and CD3+/CD8 T cell subsets in LV- and mock-transduced cells. (e and f) Relative frequency of T Central Memory (TCM), T Stem Cell Memory (TSCM), T Effector Memory (TEM) and T Effector Memory RA+ (TEMRA) in either CD3+/CD8+ (e) or CD3+/CD8 T cell subsets (f) of LV- and mock-transduced cells. Results are average SEM of n=3 independent experiments.

[0086] FIG. 7. Quantification of LV production and cell metabolism of RD3-MolPack24 and RD2-MolPack64 cells. (a) Analysis of the LV production normalized per number of cell and (b) per volume after 4 and 7 days of culture in standard T25 flasks. (c) Cell number and (d) viability at the indicated time points. (e) Dextrose consumption and lactate production at the indicated time points. (f) Viable cells in suspension at the indicated time points. **p0.01.

EXAMPLES

Example I: Derivation of Several RD3-MolPack-SIN-GFP Producer Cells

[0087] The producer cell line RD3-MolPack-SIN-GFP were obtained starting from the packaging cell line RD-MolPack disclosed in WO2012028681. Briefly RD-Molpack packaging cell line were obtained by serially loading traced HEK-293T cells with all vector genes: first HIV-1 gag, pol, rev and hygro-resistance genes by a chimeric baculo-AAV vector, thereby obtaining the so-called PK-7 clone. The rd114-tr gene was later introduced in PK-7 cells by SIN-LV delivery. Particularly, in order to obtain an efficient transduction of the rd114tr gene, PK-7 cells were transduced with VSV-G pseudo-typed transiently derived LVs containing the SIN-RD114-TR-IN and SIN-RD114-TR-IN-RRE transfer vectors (FIGS. 1b and c), to generate different RD3-MolPack packaging cells. After puromycin selection for a couple of weeks and measurement of the SIN-RD114-TR vector copy number (VCN) in several colonies, the PK-7-RD314 and PK-7-RD28 cells, containing 6 copies of the SIN-RD114-TR-IN and 12 copies of the SIN-RD114-TR-IN-RRE, respectively, were chosen as two independent RD3-MolPack packaging cells (Table 1).

TABLE-US-00001 TABLE 1 Vector copy number.sup.a of integrated genes in RD-MolPack packaging and producer cells Gag/ RD- SIN- Pol/ RD114- MolPack GFP- RD-MolPack packaging Rev Tat TR producer zeo PK-7-RD314 2 n a. 6.00 RD3-MolPack1 15.7 (RD114-TR-IN) PK-7-RD28 2 n.a. 12.0 RD3-MolPack24 48.0 (RD114-TR-IN-RRE) PK-7-RD28 2 n.a. 12.0 RD3-MolPack28 119.0 (RD114-TR-IN-RRE) PK-7-Tat7-RD19 2 6 13.0 RD2-MolPack64 2.70 (RD114-TR-IN-RRE) .sup.aThe vector copy number (VCN) was calculated by Q-PCR at least in three independent experiments using specific primers and probe sets, as reported in the supplementary material. n.a., not applicable.

[0088] The final step to obtain a prototypic 3rd-generation RD3-MolPack producer cells for SIN-LVs, consisted in introducing the SIN-TV into either PK-7-RD314 or PK-7-RD28 packaging cells. Contrary to 2nd-generation producers, in which the LTR-TV is integrated by transduction, in 3rd-generation cells, this method cannot be applied because by doing so the TV is self-inactivated at the 5LTR. The SIN-TV must be therefore integrated by stable transfection. The SIN-GFP-zeo transfer vector (TV) is disclosed in FIG. 1a. The transfer vector includes a SIN GFP cassette containing a CMV promoter followed by a U3 lentiviral 5LTR, the fragment between the splice donor and SA sites including the packaging signal and the lentiviral RRE (Dull et al. 1998); the central PolyPurine Tract (cPPT) (Follenzi et al. 2000); and the Woodchuck Hepatitis Post-Transcriptional Regulatory Element (WPRE) a Self Inactivating lentiviral 3 LTR, containing a deletion of 400 nucleotides in the 3LTR (Zuffrey et al. 1998). This construct was cloned into pBSAAVzeo plasmid, generating an intermediate SIN-GFP-zeo construct in which the TV is also flanked by two Adeno-Associated Virus (AAV)-Inverted Terminal Repeats (ITRs) (Palombo et al. 1998) and contains an SV40P-zeocin expression cassette downstream the 3U3-LTR (FIG. 1a). After Pstl cut, the SIN-GFP-zeo cassette was cloned into the into the Smal site of pGEM3.1Mlul construct thereby generating the SIN-GFP-zeo TV plasmid. The SIN-GFP-zeo TV plasmid was then linearized by Pstl to generate the SIN-GFP-zeo DNA fragment which was transfected in three independent RD-MolPack packaging cells: the 3rd-generation PK-7-RD314 and PK-7-RD28 described above and the 2nd-generation PK-7-Tat7-RD19 (i.e. RD2-MolPack) cells previously described in WO2012028681. The transfection was performed using kit Promega: ProFection Mammalian Transfection SystemCalcium Phosphate #E1200. We tested also the RD2-MolPack to verify whether Tat could somehow improve the performance of the SIN-LVs. After zeocin selection and the screening of several colonies by physical and functional titer for each cell type (Table 2), four independent producer cells were picked: 1) the RD3-MolPack1, derived from the PK-7-RD314cells carrying the SIN-RD114-TR-IN and containing 16 copies of SIN-GFP-zeo; 2-3) the RD3-MolPack24 and RD3-MolPack28, both derived from the PK-7-RD28 cells, carrying the SINRD114-TR-IN-RRE and containing 48 and 119 copies of the TV, respectively; and 4) the RD2-MolPack64, derived from the PK-7-Tat7-RD19 cells, carrying the SIN-RD114-TR-IN-RRE and containing 2.7 copies of the TV (Table 1).

TABLE-US-00002 TABLE 2 Characterization of Rd-MolPack-SIN-GFP producer cells RD3- MolPack RD3-MolPack Viability Titer p24Gag Infectivity packaging No. producer cells (%)a Titer (TU/ml) (TU/cell/day) (ng/ml) (TU/ng) PK-7-RD314 60b/47c RD3-MolPack1d 93 2.8 10.sup.4 9.2 10.sup.3 0.0056 0.002 13.0 6.20 3.8 10.sup.3 2.6 10.sup.3 PK-7-RD28 7b RD3-MolPack24e 90 3.7 10.sup.5 1.5 10.sup.5 0.074 0.039 38.3 11.1 8.5 10.sup.3 1.5 10.sup.3 PK-7-RD28 7b RD3-MolPack28d 80 2.2 10.sup.5 5.4 10.sup.4 0.043 0.011 17.5 3.40 1.2 10.sup.4 2.1 10.sup.3 RD2- MolPack packaging RD2-MolPack Viability Titer p24Gag Infectivity cells No. producer cells (%)a Titer (TU/ml) (TU/cell/day) (ng/ml) (TU/ng) PK-7-Tat7- 68b RD2-MolPack64f 91 0 10.sup.4 7.5 10.sup.3 0.056 0.009 40.4 4.4 1.8 10.sup.3 1.4 10.sup.2 RD19 aViability of the cells at the time of supernatant harvest (24 h after cell seeding). bNumber of picked-up colonies after zeocin selection. cNumber of picked-up clones after limiting dilution cloning. dMean SEM of n = 3. eMean SEM of n = 6. fMean SEM of n = 3.

Characterization of the RD3-MolPack-SIN-GFP Producers

[0089] The expression of RD3-MolPack24 and RD2-MolPack64 vector proteins was verified by Western blot analysis of cellular and virion extracts (FIG. 2). All proteins were properly translated in packaging cells, producer cells and properly processed in the derived viral particles (FIGS. 2a and 2b).

Transduction of Activated T Lymphocytes with the RD3-MolPack24

[0090] We focused our attention to the transduction of PB T lymphocytes as key cells for the treatment of hematological malignancies. We found that CD3/CD28 activated PB T lymphocytes were >92% transduced by the RD3-MolPack24 LVs at MOI=25 and >75% at MOI=1.5 either at 6 or 14 days after transduction (FIG. 3a). Accordingly, the VCN remained stable up to 14 days after transduction, ranging from 7.4 to 4 at MOI=25 and MOI=1.5, respectively (FIG. 3b).

[0091] We then compared the transduction efficiency of 36-ng p24Gag equivalents of either RD3-MolPack24 LVs (infectivity=9103 TU/ng p24Gag) or transiently derived VSV-G pseudo-typed SIN-GFP-zeo LVs (infectivity=3105 TU/ng p24Gag) on CD3/CD28 activated T cells. Six days after transduction, the percentage of GFP+ cells was almost comparable (FIG. 3c), both in CD3+/CD8 and CD3+/CD8+ subsets (FIG. 4a). In contrast, at day 14, the VCN of the GFP transgene in T cells transduced with the RD114-TR pseudo-typed LVs was three fold lower than in T cells transduced with the VSV-G pseudo-typed LVs (FIG. 3d). Most importantly, T cell memory subpopulations of the CD3+/CD8 and CD3+/CD8+ subsets were transduced with identical efficiency by the two differently pseudotyped LVs (FIG. 4c and FIG. 4d). Yet, transduction with both LVs did not affect the T-cell differentiation phenotype, which was similar to mock-transduced cells (FIG. 4b, FIG. 4e and FIG. 4f). Analogous findings were observed when the two LVs were used at the same MOI (FIGS. 5 and 6).

Cell Metabolism of RD3-MolPack24 and RD2-MolPack64 Cells

[0092] To investigate the potential manufacturing scalability of RD2- and RD3-MolPack technology, we seeded both RD2-MolPack64 and RD3-MolPack24 cells at 1.5104/cm2 in standard flask and monitored several parameters for 7 days of continuous culture (FIG. 7). Interestingly, while the two systems were comparable in terms of LV production when evaluated either as TU/cell or 10 TU/ml (FIGS. 7a and 7b), they differed in terms of metabolism and growth. The RD3-MolPack64 cells grew faster (T=25.71.4 hours vs T=32.72.9 SEM hours) (FIGS. 7c and 7e) and after 4-7 days of culture their viability decreased increasing at the same time, the number of viable cells in suspension (FIGS. 7d and 7f). Altogether, these findings indicate that RD3-MolPack system is preferable for fixed bed and hollow fiber based bioreactor, while RD2-MolPack for suspension bioreactor platform.

Medium Scale Growth of RD3-MolPack24 Cells and LV Concentration

[0093] Once established that RD3-MolPack24 cells were the best candidate for adherent growth, we optimized their culture conditions and LV concentration towards medium-large scale production. We observed almost 4-fold increment of the titer (from 4.010.sup.5 to 1.510.sup.6 TU/ml) after optimization of cell culture and harvest conditions and a slightly higher titer when the clarified supernatants were centrifuged at low rather than high speed (Table 3). We then inoculated 1.710.sup.7 cells in the multilayer HYPERFlask. From the sixth day, when the cells reached the optimal concentration for supernatant production (i.e. 2.2-2.310.sup.5 cells/cm.sup.2 equal to 410.sup.8 cells/HYPERFlask), we collected in six independent harvests, almost 3 L of supernatant containing 2.410.sup.9 total TU with a mean infectivity of 7.810.sup.38.110.sup.2 TU/ng p24Gag (Table S2). Taken together these results are encouraging for future set up of large scale manufacturing either in multi-stack vessels or in bioreactor.

TABLE-US-00003 TABLE 3 RD3-MolPack24 production in HYPERFlask Harvest Titer p24Gag Infectivity Harvest time (TU/ml).sup.a (ng/ml).sup.a (TU/ng).sup.a Volum Total TU.sup.a 72 4.9 10.sup.5 065.5 7.4 10.sup.3 500 2.4 10.sup.8 24 4.6 10.sup.5 044.5 1.0 10.sup.4 500 2.3 10.sup.8 24 5.8 10.sup.5 071.6 8.2 10.sup.3 500 2.9 10.sup.8 24 1.1 10.sup.6 134.8 8.5 10.sup.3 500 5.7 10.sup.8 24 1.3 10.sup.6 148.2 8.6 10.sup.3 500 6.4 10.sup.8 72 1.1 10.sup.6 260.6 4.1 10.sup.3 400 4.3 10.sup.8 Total 2,900 2.4 10.sup.9 .sup.aValues are representative of one of two experiments

Cells

[0094] Human embryo kidney 293T (HEK-293T) cells and its derivatives were propagated in Iscove's Modified Dulbecco's Modified Eagle Medium (IMDM) supplemented with 10% FCS and Penicillin-Streptomycin-Glutamine (PSG) (Lonza, Basel, Switzerland). CEM A3.01 T cells were grown in RPMI 1640 supplemented with 10% FCS and PSG. Human peripheral blood mononuclear cells (PBMC) were isolated from healthy donors after centrifugation on a Ficoll-Hypaque gradient (Lymphoprep; Stemcell Technologies, Vancouver, Canada) and then frozen in aliquots. PBMC were subsequently thawed and cultured in RPMI 1640 supplemented with 10% FCS, PSG and IL-7 and IL-15 (5 ng/ml, each) (Miltenyi Biotec, Bergisch Gladbach, Germany) after pre-activation with CD3/CD28 Dynabeads (LifeTechnologies Italia, Monza, Italy).

Transient and Continuous Lentiviral Vector (LV) Production, Concentration, Cell Transduction and Titer Calculation

[0095] LVs were obtained by transient co-transfection of HEK-293T cells with the following plasmids: the packaging constructs pCMV-R8.9 (3rd-gen.), the pMD.G plasmid encoding the VSV-G envelope or the SIN-RD114-TR plasmids encoding the RD114-TR envelope, and the 3rd-gen. (SIN-GFP) transfer vector (TV). Supernatants were harvested 48 hours after transfection.

[0096] Continuous LV production was routinely obtained by cultivating RD-MolPack cells in different cell culture devices: standard flasks, a small disposable bioreactor and Hyperflasks. To evaluate the productivity of the RD-MolPack cells, T75 flasks were inoculated with 2.510.sup.6/cm2 in 0.5 ml/cm2 IMDM supplemented with 10% FBS and PSG and after 24 hours supernatant was harvested. RD-MolPack LVs were also produced by inoculating 2.510.sup.7 RD-MolPack cells in the CELLine AD 1000 bioreactor (INTEGRA Biosciences AG, CH) and replacing the cell compartment medium (15-ml IMDM+10% FCS) every 48 hours, corresponding to a single harvest, whereas the medium compartment (1000-ml IMDM+1% FCS) weekly for up to 3 months. In selected experiments, RD-MolPack cells were cultured in Corning HYPERFlask (Corning Inc. Life Science, Lowell, Mass.) by inoculating 1.710.sup.7 cells/flask and collecting the supernatant after 6 days, when the total number of cells reached approximately 410.sup.8. In all production systems, the harvested supernatant was clarified by 0.45-m filtration and stored at 80 C. until use. When indicated, the supernatants were concentrated 100-fold by either low speed (3,761g at +4 C. for 16 hours in a Multifuge 32-R centrifuge) or high speed centrifugation (50,000g at +4 C. for 2 hours in a Beckman L-80 ultracentrifuge). The viral pellets were resuspended in IMDM medium supplemented with 10% FBS and frozen at 80 C. for deferred use. Transduction of CEM A3.01 cell line and activated T lymphocytes was carried out by one cycle of spinoculation at 1,024g for 2 hours at 37 C. in the presence of polybrene (8 g/ml) (Sigma-Aldrich, St Louis, Mo.); transduction efficiency was monitored by flow cytometry analysis (FACS Canto II Instrument, BD Bioscience, San Jose, Calif.) of GFP (SIN-GFP) by the DIVA software (BD Bioscience). LV titer was calculated on CEM A3.01 cells using the 5-25% range of transduction efficiency as previously described [Porcellini et al. 2010]. Physical particles (pp) production was estimated by measuring the p24Gag released in the supernatants by the Alliance HIV-1 p24 Antigen ELISA kit (Perkin Elmer, Inc. Waltham, Mass.) following manufacturer's instructions. It is assumed that 1 ng of p24Gag corresponds to 110.sup.7 pp.

Western Blot Assay

[0097] Cellular and viral proteins, the latter derived from isolated cell-free VLPs or LVs, were prepared as previously described and then separated by SDS-PAGE on 4-15% Mini-PROTEAN TGX precast gels (Biorad, Hercules, Calif., USA). The primary antibodies used were the following: anti-HIV serum, obtained from an AIDS patient, and kindly donated by G. Poli (OSR, Milano, Italy) diluted 1:1,000; the anti-TM RD114-TR rabbit serum, kindly provided by F. L. Cosset (INSERM, France) diluted 1:1,000; anti-Rev mouse antibody, from Santa Cruz Biotechnology (sc-69730), diluted 1:500; anti-Tat mouse antibody, obtained from the NIH AIDS Research and Reference Reagent Program (USA), diluted 1:500; anti-actin rabbit antibody, (A 2066) (Sigma-Aldrich), diluted 1:2,500. The secondary HRP-linked antibodies used were: anti-human (NA933V) and anti-rabbit (NA934V) (GE Healthcare, Europe GmbH) diluted 1:5,000; anti-mouse (A2066) (Sigma-Aldrich) diluted 1:10,000. For the chemiluminescence reaction we used the ECL-Western Blotting Detection Reagents (RPMN2106) (GE Healthcare).

RD3-MolPack24 and RD2-MolPack64 Cell Metabolism

[0098] RD3-MolPack24 and RD2-MolPack64 cells were seeded at 1.5104 cells/cm2 density in T25 flasks. Cells were counted daily for a week and at each time point, supernatants were harvested to measure glucose consumption and lactate production by the YSI 2700 SELECT Biochemistry Analyzer (YSI, Yellow Springs, Ohio) and the functional and physical titers as described above.