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Comparative analysis of lentiviral vectors and modular protein nanovectors for traumatic brain injury gene therapy.

Negro-Demontel ML, Saccardo P, Giacomini C, Yáñez-Muñoz RJ, Ferrer-Miralles N, Vazquez E, Villaverde A, Peluffo H - Mol Ther Methods Clin Dev (2014)

Bottom Line: While lentiviral vectors showed GFP protein 1 day after TBI and increased expression at 14 days, nanovectors showed stable and lower GFP transgene expression from 1 to 14 days.No toxicity after TBI by any of the vectors was observed as determined by resulting levels of IL-1β or using neurological sticky tape test.In fact, both vector types induced functional improvement per se.

View Article: PubMed Central - PubMed

Affiliation: Neuroinflammation and Gene Therapy Laboratory, Institut Pasteur de Montevideo , Montevideo, Uruguay ; Departmento de Histología y Embriología, Facultad de Medicina, UDELAR , Montevideo, Uruguay.

ABSTRACT
Traumatic brain injury (TBI) remains as one of the leading causes of mortality and morbidity worldwide and there are no effective treatments currently available. Gene therapy applications have emerged as important alternatives for the treatment of diverse nervous system injuries. New strategies are evolving with the notion that each particular pathological condition may require a specific vector. Moreover, the lack of detailed comparative studies between different vectors under similar conditions hampers the selection of an ideal vector for a given pathological condition. The potential use of lentiviral vectors versus several modular protein-based nanovectors was compared using a controlled cortical impact model of TBI under the same gene therapy conditions. We show that variables such as protein/DNA ratio, incubation volume, and presence of serum or chloroquine in the transfection medium impact on both nanovector formation and transfection efficiency in vitro. While lentiviral vectors showed GFP protein 1 day after TBI and increased expression at 14 days, nanovectors showed stable and lower GFP transgene expression from 1 to 14 days. No toxicity after TBI by any of the vectors was observed as determined by resulting levels of IL-1β or using neurological sticky tape test. In fact, both vector types induced functional improvement per se.

No MeSH data available.


Related in: MedlinePlus

In vitro transfection and transduction efficiency of vectors. HEK293T cells were incubated for 4 hours with DNA, lipofectamine (LIPO), or modular protein nanovectors at different protein/DNA mass ratios (ratios from 2.5 to 7.5 are shown), and transfection efficiencies (a) and relative fluorescence intensity per cell (b) were quantified 1 day later by flow cytometry. DNA concentrations used (2 μg/well, 2.4 × 1011 copies of gfp gene) were the same in all cases so as to compare transfection efficiencies. HNRK and HKRN vectors were purified from insoluble bacterial fractions and compared with HNRK obtained from soluble bacterial fractions (HNRKs). In parallel experiments, HEK293T cells were incubated with lentiviral vectors at different multiplicities of infections (MOIs) (at MOI 1, there are 1.4 × 106 to 1.4 × 107 copies of the gfp gene) and transduction efficiencies (c) and relative fluorescence intensity per cell (d) were quantified at 1 and 3 days post-transduction (dpt). a, b: *P < 0.05 when compared with NLSCt, DNA, or HNRKs; c, d: *P < 0.05 when compared with DNA.
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fig1: In vitro transfection and transduction efficiency of vectors. HEK293T cells were incubated for 4 hours with DNA, lipofectamine (LIPO), or modular protein nanovectors at different protein/DNA mass ratios (ratios from 2.5 to 7.5 are shown), and transfection efficiencies (a) and relative fluorescence intensity per cell (b) were quantified 1 day later by flow cytometry. DNA concentrations used (2 μg/well, 2.4 × 1011 copies of gfp gene) were the same in all cases so as to compare transfection efficiencies. HNRK and HKRN vectors were purified from insoluble bacterial fractions and compared with HNRK obtained from soluble bacterial fractions (HNRKs). In parallel experiments, HEK293T cells were incubated with lentiviral vectors at different multiplicities of infections (MOIs) (at MOI 1, there are 1.4 × 106 to 1.4 × 107 copies of the gfp gene) and transduction efficiencies (c) and relative fluorescence intensity per cell (d) were quantified at 1 and 3 days post-transduction (dpt). a, b: *P < 0.05 when compared with NLSCt, DNA, or HNRKs; c, d: *P < 0.05 when compared with DNA.

Mentions: Modular protein nanovectors HNRK and HKRN were incubated with plasmid DNA at room temperature, to generate self-organized nanoparticles of ca. 80 nm.10 Since different protein/DNA mass ratios can result in different transfection efficiencies,10 we analyzed transfection efficiency of HNRK and HKRN vectors formed at different protein/DNA mass ratios. This was done taking into account that protein/DNA mass ratios of 2.5 and 1.5 of HNRK and HKRN, respectively, result in complete retardation of total DNA in the sample in gel retardation assays.10 HEK293T cells were incubated with nanovectors loaded with 2 μg of a GFP-expressing plasmid transcriptionally controlled by a CMV promoter. Percentage of cells transfected and the relative fluorescence intensity/cell were analyzed 24 hours after transfection. The HNRK and HKRN vectors purified from bacterial inclusion bodies had increased transfection efficiency when compared to the same vectors purified from soluble bacterial fractions (HNRKs), or when compared to the similar vector NLSCt or to naked DNA (Figure 1a). Increased protein/DNA mass ratios in nanocomplexes formed resulted in increased transfection efficiency with optimal ratios of 4.5–5 (Figure 1a), and declining efficiency at higher ratios thereafter (not shown). Interestingly, although a higher percentage of cells were transfected with lipofectamine than with HNRK or HKRN nanocomplexes, relative fluorescence intensity/cell was similar for the three groups at 4.5–5 protein/DNA mass ratios (Figure 1b). Also, there was no decrease in cell viability after treatment with nanocomplexes (not shown). We next transduced HEK293T cells with third-generation lentiviral vectors pseudotyped with VSV envelope protein. These vectors carried the same expression cassette used in the nanovector transfection assay, and were analyzed 1 and 3 days post-transfection/transduction (dpt) for percentage of cells transduced and for relative fluorescence intensity/cell. Transfection of cells with HNRK or HKRN resulted in maximal transgene expression at 24 hours (not shown), however and as previously reported lentiviral vectors required at least 3 days for full transgene expression (Figure 1c,d).16 Surprisingly, GFP was detected as early as 1 dpt for all multiplicities of infection (MOIs) tested. Cell toxicity was observed at 3 dpt for the highest MOI tested (i.e., MOI = 4.4, data not shown). HEK293T cell transduction efficiency, however, was higher for lentiviral vectors (at MOI 1, there are 1.4 × 106 to 1.4 × 107 copies of the gfp gene in transduced cells according to QPCR data derived calculations in ref. 33) compared with the nanovectors (the 2 μg DNA used per well corresponds to 2.4 × 1011 copies of gfp gene) in terms of percentage of transduced cells (Figure 1a–d). In terms of relative fluorescence intensity/cell, qualitative microscopic observations confirmed the increased intensity after lentiviral transduction when compared to nanovector transfection. Comparative quantitative data are shown in Table 1.


Comparative analysis of lentiviral vectors and modular protein nanovectors for traumatic brain injury gene therapy.

Negro-Demontel ML, Saccardo P, Giacomini C, Yáñez-Muñoz RJ, Ferrer-Miralles N, Vazquez E, Villaverde A, Peluffo H - Mol Ther Methods Clin Dev (2014)

In vitro transfection and transduction efficiency of vectors. HEK293T cells were incubated for 4 hours with DNA, lipofectamine (LIPO), or modular protein nanovectors at different protein/DNA mass ratios (ratios from 2.5 to 7.5 are shown), and transfection efficiencies (a) and relative fluorescence intensity per cell (b) were quantified 1 day later by flow cytometry. DNA concentrations used (2 μg/well, 2.4 × 1011 copies of gfp gene) were the same in all cases so as to compare transfection efficiencies. HNRK and HKRN vectors were purified from insoluble bacterial fractions and compared with HNRK obtained from soluble bacterial fractions (HNRKs). In parallel experiments, HEK293T cells were incubated with lentiviral vectors at different multiplicities of infections (MOIs) (at MOI 1, there are 1.4 × 106 to 1.4 × 107 copies of the gfp gene) and transduction efficiencies (c) and relative fluorescence intensity per cell (d) were quantified at 1 and 3 days post-transduction (dpt). a, b: *P < 0.05 when compared with NLSCt, DNA, or HNRKs; c, d: *P < 0.05 when compared with DNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig1: In vitro transfection and transduction efficiency of vectors. HEK293T cells were incubated for 4 hours with DNA, lipofectamine (LIPO), or modular protein nanovectors at different protein/DNA mass ratios (ratios from 2.5 to 7.5 are shown), and transfection efficiencies (a) and relative fluorescence intensity per cell (b) were quantified 1 day later by flow cytometry. DNA concentrations used (2 μg/well, 2.4 × 1011 copies of gfp gene) were the same in all cases so as to compare transfection efficiencies. HNRK and HKRN vectors were purified from insoluble bacterial fractions and compared with HNRK obtained from soluble bacterial fractions (HNRKs). In parallel experiments, HEK293T cells were incubated with lentiviral vectors at different multiplicities of infections (MOIs) (at MOI 1, there are 1.4 × 106 to 1.4 × 107 copies of the gfp gene) and transduction efficiencies (c) and relative fluorescence intensity per cell (d) were quantified at 1 and 3 days post-transduction (dpt). a, b: *P < 0.05 when compared with NLSCt, DNA, or HNRKs; c, d: *P < 0.05 when compared with DNA.
Mentions: Modular protein nanovectors HNRK and HKRN were incubated with plasmid DNA at room temperature, to generate self-organized nanoparticles of ca. 80 nm.10 Since different protein/DNA mass ratios can result in different transfection efficiencies,10 we analyzed transfection efficiency of HNRK and HKRN vectors formed at different protein/DNA mass ratios. This was done taking into account that protein/DNA mass ratios of 2.5 and 1.5 of HNRK and HKRN, respectively, result in complete retardation of total DNA in the sample in gel retardation assays.10 HEK293T cells were incubated with nanovectors loaded with 2 μg of a GFP-expressing plasmid transcriptionally controlled by a CMV promoter. Percentage of cells transfected and the relative fluorescence intensity/cell were analyzed 24 hours after transfection. The HNRK and HKRN vectors purified from bacterial inclusion bodies had increased transfection efficiency when compared to the same vectors purified from soluble bacterial fractions (HNRKs), or when compared to the similar vector NLSCt or to naked DNA (Figure 1a). Increased protein/DNA mass ratios in nanocomplexes formed resulted in increased transfection efficiency with optimal ratios of 4.5–5 (Figure 1a), and declining efficiency at higher ratios thereafter (not shown). Interestingly, although a higher percentage of cells were transfected with lipofectamine than with HNRK or HKRN nanocomplexes, relative fluorescence intensity/cell was similar for the three groups at 4.5–5 protein/DNA mass ratios (Figure 1b). Also, there was no decrease in cell viability after treatment with nanocomplexes (not shown). We next transduced HEK293T cells with third-generation lentiviral vectors pseudotyped with VSV envelope protein. These vectors carried the same expression cassette used in the nanovector transfection assay, and were analyzed 1 and 3 days post-transfection/transduction (dpt) for percentage of cells transduced and for relative fluorescence intensity/cell. Transfection of cells with HNRK or HKRN resulted in maximal transgene expression at 24 hours (not shown), however and as previously reported lentiviral vectors required at least 3 days for full transgene expression (Figure 1c,d).16 Surprisingly, GFP was detected as early as 1 dpt for all multiplicities of infection (MOIs) tested. Cell toxicity was observed at 3 dpt for the highest MOI tested (i.e., MOI = 4.4, data not shown). HEK293T cell transduction efficiency, however, was higher for lentiviral vectors (at MOI 1, there are 1.4 × 106 to 1.4 × 107 copies of the gfp gene in transduced cells according to QPCR data derived calculations in ref. 33) compared with the nanovectors (the 2 μg DNA used per well corresponds to 2.4 × 1011 copies of gfp gene) in terms of percentage of transduced cells (Figure 1a–d). In terms of relative fluorescence intensity/cell, qualitative microscopic observations confirmed the increased intensity after lentiviral transduction when compared to nanovector transfection. Comparative quantitative data are shown in Table 1.

Bottom Line: While lentiviral vectors showed GFP protein 1 day after TBI and increased expression at 14 days, nanovectors showed stable and lower GFP transgene expression from 1 to 14 days.No toxicity after TBI by any of the vectors was observed as determined by resulting levels of IL-1β or using neurological sticky tape test.In fact, both vector types induced functional improvement per se.

View Article: PubMed Central - PubMed

Affiliation: Neuroinflammation and Gene Therapy Laboratory, Institut Pasteur de Montevideo , Montevideo, Uruguay ; Departmento de Histología y Embriología, Facultad de Medicina, UDELAR , Montevideo, Uruguay.

ABSTRACT
Traumatic brain injury (TBI) remains as one of the leading causes of mortality and morbidity worldwide and there are no effective treatments currently available. Gene therapy applications have emerged as important alternatives for the treatment of diverse nervous system injuries. New strategies are evolving with the notion that each particular pathological condition may require a specific vector. Moreover, the lack of detailed comparative studies between different vectors under similar conditions hampers the selection of an ideal vector for a given pathological condition. The potential use of lentiviral vectors versus several modular protein-based nanovectors was compared using a controlled cortical impact model of TBI under the same gene therapy conditions. We show that variables such as protein/DNA ratio, incubation volume, and presence of serum or chloroquine in the transfection medium impact on both nanovector formation and transfection efficiency in vitro. While lentiviral vectors showed GFP protein 1 day after TBI and increased expression at 14 days, nanovectors showed stable and lower GFP transgene expression from 1 to 14 days. No toxicity after TBI by any of the vectors was observed as determined by resulting levels of IL-1β or using neurological sticky tape test. In fact, both vector types induced functional improvement per se.

No MeSH data available.


Related in: MedlinePlus