Efficient Non-Invasive Plasmid-DNA Administration into Tibialis Cranialis Muscle of “Little” Mice

Cecchi, Claudia R.; Higuti, Eliza; Lima, Eliana R.; Vieira, Daniel Perez; Squair, Peterson Lima; Peroni, Cibele N.; Bartolini, Paolo

doi:10.2174/1566524017666170721152119

Cited by 2 publications

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“…The High Voltage/Low Voltage (HV/LV) electrotransfer protocol, based on one 800 V/cm pulse of 100 µs followed by one 400 ms pulse of 100 V/cm [11,13], was compared to a new protocol based on three 375 V/cm pulses of 25 ms each [14,15], providing the results shown in Figure 1A, where the advantage of injecting a volume of 20 µL was also confirmed. The higher efficiency obtained with the 375 V/cm protocol led us to adopt this strategy for the subsequent experiments.…”

Section: Resultsmentioning

confidence: 99%

“…The higher efficiency obtained with the 375 V/cm protocol led us to adopt this strategy for the subsequent experiments. Moreover, because we had demonstrated the advantage of transfecting the tibialis cranialis muscle rather than the exposed quadriceps [11] this modification was also maintained. electroporation was reported in 2008 for an interleukin-12 plasmid delivered into the tumors of patients suffering metastatic melanoma [7].…”

Section: Resultsmentioning

confidence: 99%

“…Although 50 μg of DNA was routinely used in each injection [4,5,[9][10][11], it was necessary to confirm whether this was still the optimum amount of DNA to be administrated under the new conditions. As shown in Figure 1B, the new conditions had no effect on the optimum amount of DNA and a slightly higher transfection efficiency, though statistically non-significant, was still obtained with 50 μg DNA per injection.…”

Section: Resultsmentioning

confidence: 99%

“…This result is presented in Figure 2A, where 14.7 ± 3.7 ng/mL vs. 0.5 ± 0.2 ng/mL for saline (n = 3 for each group; p < 0.001) were obtained when the approach of two injections into each tibialis cranialis muscle was employed, in comparison with two injections into a single tibialis cranialis muscle (5.9 ± 2.7 ng/mL, p < 0.02, n = 3). Even higher levels of up to 21.1 ± 1.0 ng hGH/mL vs. 1.1 ± 0.1 Although 50 µg of DNA was routinely used in each injection [4,5,[9][10][11], it was necessary to confirm whether this was still the optimum amount of DNA to be administrated under the new conditions. As shown in Figure 1B, the new conditions had no effect on the optimum amount of DNA and a slightly higher transfection efficiency, though statistically non-significant, was still obtained with 50 µg DNA per injection.…”

Section: Resultsmentioning

confidence: 99%

“…To set up gene therapy protocols based on growth hormone plasmid DNA administration, we have utilized immunodeficient (lit/scid) or immunocompetent (lit/lit) dwarf mice in order to investigate electrotransfer of human (hGH) or mouse (mGH) genes into muscle [4,5,[9][10][11]. In these studies, we routinely encounter better results with the administration of hGH DNA to immunodeficient dwarf mice.…”

Section: Introductionmentioning

confidence: 99%

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Optimization of Mouse Growth Hormone Plasmid DNA Electrotransfer into Tibialis Cranialis Muscle of “Little” Mice

et al. 2020

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Previous non-viral gene therapy was directed towards two animal models of dwarfism: Immunodeficient (lit/scid) and immunocompetent (lit/lit) dwarf mice. The former, based on hGH DNA administration into muscle, performed better, while the latter, a homologous model based on mGH DNA, was less efficient, though recommended as useful for pre-clinical assays. We have now improved the growth parameters aiming at a complete recovery of the lit/lit phenotype. Electrotransfer was based on three pulses of 375 V/cm of 25 ms each, after mGH-DNA administration into two sites of each non-exposed tibialis cranialis muscle. A 36-day bioassay, performed using 60-day old lit/lit mice, provided the highest GH circulatory levels we have ever obtained for GH non-viral gene therapy: 14.7 ± 3.7 ng mGH/mL. These levels, at the end of the experiment, were 8.5 ± 2.3 ng/mL, i.e., significantly higher than those of the positive control (4.5 ± 1.5 ng/mL). The catch-up growth reached 40.9% for body weight, 38.2% for body length and 82.6%–76.9% for femur length. The catch-up in terms of the mIGF-1 levels remained low, increasing from the previous value of 5.9% to the actual 8.5%. Although a complete phenotypic recovery was not obtained, it should be possible starting with much younger animals and/or increasing the number of injection sites.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Optimization of Mouse Growth Hormone Plasmid DNA Electrotransfer into Tibialis Cranialis Muscle of “Little” Mice

et al. 2020

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Isolation and Characterization of the Arapaima gigas Growth Hormone (ag-GH) cDNA and Three-Dimensional Modeling of This Hormone in Comparison with the Human Hormone (hGH)

et al. 2023

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In a previous work, the common gonadotrophic hormone α-subunit (ag-GTHα), the ag-FSH β- and ag-LH β-subunit cDNAs, were isolated and characterized by our research group from A. gigas pituitaries, while a preliminary synthesis of ag-FSH was also carried out in human embryonic kidney 293 (HEK293) cells. In the present work, the cDNA sequence encoding the ag-growth hormone (ag-GH) has also been isolated from the same giant Arapaimidae Amazonian fish. The ag-GH consists of 208 amino acids with a putative 23 amino acid signal peptide and a 185 amino acid mature peptide. The highest identity, based on the amino acid sequences, was found with the Elopiformes (82.0%), followed by Anguilliformes (79.7%) and Acipenseriformes (74.5%). The identity with the corresponding human GH (hGH) amino acid sequence is remarkable (44.8%), and the two disulfide bonds present in both sequences were perfectly conserved. Three-dimensional (3D) models of ag-GH, in comparison with hGH, were generated using the threading modeling method followed by molecular dynamics. Our simulations suggest that the two proteins have similar structural properties without major conformational changes under the simulated conditions, even though they are separated from each other by a >100 Myr evolutionary period (1 Myr = 1 million years). The sequence found will be used for the biotechnological synthesis of ag-GH while the ag-GH cDNA obtained will be utilized for preliminary Gene Therapy studies.

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Efficient Non-Invasive Plasmid-DNA Administration into Tibialis Cranialis Muscle of “Little” Mice

Abstract: These results show that hGH-DNA administration into non-exposed tibialis muscle followed by the new HV/LV electrotransfer protocol was an equally efficient, less traumatic treatment, much more suitable for pre-clinical testing than administration into exposed quadriceps.

Cited by 2 publications

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Optimization of Mouse Growth Hormone Plasmid DNA Electrotransfer into Tibialis Cranialis Muscle of “Little” Mice

Optimization of Mouse Growth Hormone Plasmid DNA Electrotransfer into Tibialis Cranialis Muscle of “Little” Mice

Isolation and Characterization of the Arapaima gigas Growth Hormone (ag-GH) cDNA and Three-Dimensional Modeling of This Hormone in Comparison with the Human Hormone (hGH)

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