Optimization of Mouse Growth Hormone Plasmid DNA Electrotransfer into Tibialis Cranialis Muscle of "Little" Mice.

Eliana Rosa Lima,Cibele Nunes Peroni,Gustavo Protasio Pacheco De Jesus,Claudia Regina Cecchi,Eliza Higuti,Enio Aparecido Zacarias,Paolo Bartolini,Alissandra Moura Gomes

doi:10.3390/molecules25215034

Abstract

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.

Highlights

The non-viral gene delivery system using plasmid DNA that in most cases is injected directly into certain tissues, muscle, produces significant levels of gene expression, typically lower than those achieved with viral vectors
The newly set up electroporation and assay conditions provided, as far as we know, the highest circulatory levels of hGH in lit/scid and of mGH in lit/lit ever reported for GH non-viral gene therapy and will greatly facilitate the utilization of the homologous model for pre-clinical assay
Concerning the initial electrotransfer protocol (Figure 1A), it was considered to be more practical and more efficient to carry out this first optimization in a 3-day assay, using a single hGH DNA plasmid administration to lit/scid mice

Summary

Introduction

The non-viral gene delivery system using plasmid DNA that in most cases is injected directly into certain tissues, muscle, produces significant levels of gene expression, typically lower than those achieved with viral vectors. This type of gene therapy is among the most frequently used in clinical trials, representing 16.6% of the worldwide protocols in 2017, close behind adenovirus (20.1%) and retrovirus (17.9%) administrations [1]. The first human trial of gene transfer using electroporation was Molecules 2020, 25, 5034; doi:10.3390/molecules25215034 www.mdpi.com/journal/molecules electroporation was reported infor for an interleukin-12 plasmid delivered into the tumors of electroporation was reported interleukin-12 plasmid delivered into the tumors electroporation was reported in an interleukin-12 plasmid delivered into the tumors of electroporation was reported inin forfor anan interleukin-12 plasmid delivered into the tumors ofof patients suffering metastatic melanoma [7].

Methods

Results

Discussion

Conclusion