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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: Traumatic brain injury (TBI) remains as one of the leading causes of mortality and morbidity worldwide and there are no effective treatments currently available.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

Effect of incubation volume and medium on the protein nanovector complex size and transfection efficiency. HNRK and HKRN modular protein nanovectors were allowed to self-assembly during a 20-minute incubation of HNRK and HKRN proteins with plasmid DNA in either 200 μl OPTIPRO medium or 30 μl PBS. Assembled nanovectors were then used to transfect HEK293T cells and transfection efficiencies were quantified 1 day later by flow cytometry (a, b). HNRK nanovector stability was analyzed after self-assembly in 200 μl OPTIPRO medium or 30 μl phosphate-buffered saline, and for 20 minutes to 5 hours incubation periods. The products formed were analyzed by dynamic light scattering (c) or transmission electron microscopy (d) as indicated. Transfection efficiency of HNRK nanovector after self-assembling in 200 μl OPTIPRO medium for 20 minutes to 5 hours was analyzed by flow cytometry 1 day after transfection (100% represents transfection efficiency for nanovectors formed in the 20 minutes incubation time). *P < 0.05 when compared to vectors allowed to self-assembly in 200 μl OPTIPRO.
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fig3: Effect of incubation volume and medium on the protein nanovector complex size and transfection efficiency. HNRK and HKRN modular protein nanovectors were allowed to self-assembly during a 20-minute incubation of HNRK and HKRN proteins with plasmid DNA in either 200 μl OPTIPRO medium or 30 μl PBS. Assembled nanovectors were then used to transfect HEK293T cells and transfection efficiencies were quantified 1 day later by flow cytometry (a, b). HNRK nanovector stability was analyzed after self-assembly in 200 μl OPTIPRO medium or 30 μl phosphate-buffered saline, and for 20 minutes to 5 hours incubation periods. The products formed were analyzed by dynamic light scattering (c) or transmission electron microscopy (d) as indicated. Transfection efficiency of HNRK nanovector after self-assembling in 200 μl OPTIPRO medium for 20 minutes to 5 hours was analyzed by flow cytometry 1 day after transfection (100% represents transfection efficiency for nanovectors formed in the 20 minutes incubation time). *P < 0.05 when compared to vectors allowed to self-assembly in 200 μl OPTIPRO.

Mentions: Another limiting factor for in vivo administration of these vectors is the volume that can be injected into CNS parenchyma due to very limited extracellular space. Thus, we evaluated the effect of nanocomplex formation in a reduced volume. A reduction in nanocomplex formation volume, i.e., from 200 to 30 µl OPTIPRO medium, however, resulted in 52 and 42% decreases in HEK293T cell transfection efficiency for HNRK and HKRN nanovectors, respectively (Figure 3a,b). Another important variable that can affect nanocomplex formation and transfection efficiency is protein/DNA incubation time. Analysis of HNRK/DNA nanocomplex particles formed using dynamic light scattering (DLS) and transmission electron microscopy (TEM) indicated particle size tended to be less variable when produced in 200 μl OPTIPRO medium compared with being produced in 30 μl phosphate-buffered saline (PBS), the vehicle used for in vivo administration of vectors (Figure 3c,d). There were no significant differences in nanocomplex formation with either 20 minutes or 5 hours incubation times for either condition (200 µl OPTIPRO or 30 μl PBS). Accordingly, no differences were observed in HEK293T cell transfection efficiency of HNRK/pDNA nanovectors formed in 200 μl OPTIPRO over 20 minutes versus those formed over 5 hours (Figure 3e). Nanocomplex size observed by TEM and hydrodynamic diameter observed by DLS differed. While TEM images showed particles to be between 20–200 nm, DLS indicated particles to be between 1–2 μm suggesting clustering or aggregation of particles.


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)

Effect of incubation volume and medium on the protein nanovector complex size and transfection efficiency. HNRK and HKRN modular protein nanovectors were allowed to self-assembly during a 20-minute incubation of HNRK and HKRN proteins with plasmid DNA in either 200 μl OPTIPRO medium or 30 μl PBS. Assembled nanovectors were then used to transfect HEK293T cells and transfection efficiencies were quantified 1 day later by flow cytometry (a, b). HNRK nanovector stability was analyzed after self-assembly in 200 μl OPTIPRO medium or 30 μl phosphate-buffered saline, and for 20 minutes to 5 hours incubation periods. The products formed were analyzed by dynamic light scattering (c) or transmission electron microscopy (d) as indicated. Transfection efficiency of HNRK nanovector after self-assembling in 200 μl OPTIPRO medium for 20 minutes to 5 hours was analyzed by flow cytometry 1 day after transfection (100% represents transfection efficiency for nanovectors formed in the 20 minutes incubation time). *P < 0.05 when compared to vectors allowed to self-assembly in 200 μl OPTIPRO.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig3: Effect of incubation volume and medium on the protein nanovector complex size and transfection efficiency. HNRK and HKRN modular protein nanovectors were allowed to self-assembly during a 20-minute incubation of HNRK and HKRN proteins with plasmid DNA in either 200 μl OPTIPRO medium or 30 μl PBS. Assembled nanovectors were then used to transfect HEK293T cells and transfection efficiencies were quantified 1 day later by flow cytometry (a, b). HNRK nanovector stability was analyzed after self-assembly in 200 μl OPTIPRO medium or 30 μl phosphate-buffered saline, and for 20 minutes to 5 hours incubation periods. The products formed were analyzed by dynamic light scattering (c) or transmission electron microscopy (d) as indicated. Transfection efficiency of HNRK nanovector after self-assembling in 200 μl OPTIPRO medium for 20 minutes to 5 hours was analyzed by flow cytometry 1 day after transfection (100% represents transfection efficiency for nanovectors formed in the 20 minutes incubation time). *P < 0.05 when compared to vectors allowed to self-assembly in 200 μl OPTIPRO.
Mentions: Another limiting factor for in vivo administration of these vectors is the volume that can be injected into CNS parenchyma due to very limited extracellular space. Thus, we evaluated the effect of nanocomplex formation in a reduced volume. A reduction in nanocomplex formation volume, i.e., from 200 to 30 µl OPTIPRO medium, however, resulted in 52 and 42% decreases in HEK293T cell transfection efficiency for HNRK and HKRN nanovectors, respectively (Figure 3a,b). Another important variable that can affect nanocomplex formation and transfection efficiency is protein/DNA incubation time. Analysis of HNRK/DNA nanocomplex particles formed using dynamic light scattering (DLS) and transmission electron microscopy (TEM) indicated particle size tended to be less variable when produced in 200 μl OPTIPRO medium compared with being produced in 30 μl phosphate-buffered saline (PBS), the vehicle used for in vivo administration of vectors (Figure 3c,d). There were no significant differences in nanocomplex formation with either 20 minutes or 5 hours incubation times for either condition (200 µl OPTIPRO or 30 μl PBS). Accordingly, no differences were observed in HEK293T cell transfection efficiency of HNRK/pDNA nanovectors formed in 200 μl OPTIPRO over 20 minutes versus those formed over 5 hours (Figure 3e). Nanocomplex size observed by TEM and hydrodynamic diameter observed by DLS differed. While TEM images showed particles to be between 20–200 nm, DLS indicated particles to be between 1–2 μm suggesting clustering or aggregation of particles.

Bottom Line: Traumatic brain injury (TBI) remains as one of the leading causes of mortality and morbidity worldwide and there are no effective treatments currently available.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