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

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.


Introduction
The non-viral gene delivery system using plasmid DNA that in most cases is injected directly into certain tissues, particularly muscle, produces significant levels of gene expression, although typically lower than those achieved with viral vectors. Nonetheless, 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]. Non-viral methods have several advantages compared with viral vectors, including relative safety, the ability to transfer much larger genes, less toxicity and easier DNA preparation [2]. Its relatively low transfection efficiency and poor transgene expression can be substantially increased when plasmid DNA administration is followed by electroporation [1][2][3][4][5][6]. The first human trial of gene transfer using electroporation was reported in 2008 for an interleukin-12 plasmid delivered into the tumors of patients suffering metastatic melanoma [7]. Close to 90 clinical trials involving electrotransfer have been performed subsequently and the technique is considered to be relatively inexpensive, flexible and safe because it does not introduce any additional chemicals or viruses into cells [8]. However, as far as we know, the utilization of naked DNA is still not at the clinical level for systemic diseases [5].
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. In previous work, the injection of the hGH gene into the tibialis cranialis (TA) muscle of 40 day old lit/scid mice provided a high catch-up growth of 76% for femur, 36% for nose-to-tail length and 39% for tail length [5]. Further studies on the different in vivo expressions of either hGH or mGH in these mice models could also take advantage of a recent review in which partially humanized transgenic mice were able to co-express hGH and mGH, offering a unique opportunity of investigation [12].
In the present study, we have carried out an initial optimization of this methodology to improve the transfection efficiency in the homologous model based on genomic mGH DNA electrotransfer into the tibialis muscle of immunocompetent lit/lit mice, taking into account the recommendation of the use of this model for setting up preclinical testing of non-viral gene therapy.

Results
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. 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]. Close to 90 clinical trials involving electrotransfer have been performed subsequently and the technique is considered to be relatively inexpensive, flexible and safe because it does not introduce any additional chemicals or viruses into cells [8]. However, as far as we know, the utilization of naked DNA is still not at the clinical level for systemic diseases [5].
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. In previous work, the injection of the hGH gene into the tibialis cranialis (TA) muscle of 40 day old lit/scid mice provided a high catch-up growth of 76% for femur, 36% for nose-to-tail length and 39% for tail length [5]. Further studies on the different in vivo expressions of either hGH or mGH in these mice models could also take advantage of a recent review in which partially humanized transgenic mice were able to co-express hGH and mGH, offering a unique opportunity of investigation [12].
In the present study, we have carried out an initial optimization of this methodology to improve the transfection efficiency in the homologous model based on genomic mGH DNA electrotransfer into the tibialis muscle of immunocompetent lit/lit mice, taking into account the recommendation of the use of this model for setting up preclinical testing of non-viral gene therapy.

Results
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. Moreover, because we had demonstrated the advantage of transfecting the tibialis cranialis muscle rather than the exposed quadriceps [11] this modification was also maintained.
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.  electroporation was reported in 2008 for an interleukin-12 plasmid delivered into the tumors of patients suffering metastatic melanoma [7]. Close to 90 clinical trials involving electrotransfer have been performed subsequently and the technique is considered to be relatively inexpensive, flexible and safe because it does not introduce any additional chemicals or viruses into cells [8]. However, as far as we know, the utilization of naked DNA is still not at the clinical level for systemic diseases [5].
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. In previous work, the injection of the hGH gene into the tibialis cranialis (TA) muscle of 40 day old lit/scid mice provided a high catch-up growth of 76% for femur, 36% for nose-to-tail length and 39% for tail length [5]. Further studies on the different in vivo expressions of either hGH or mGH in these mice models could also take advantage of a recent review in which partially humanized transgenic mice were able to co-express hGH and mGH, offering a unique opportunity of investigation [12].
In the present study, we have carried out an initial optimization of this methodology to improve the transfection efficiency in the homologous model based on genomic mGH DNA electrotransfer into the tibialis muscle of immunocompetent lit/lit mice, taking into account the recommendation of the use of this model for setting up preclinical testing of non-viral gene therapy.

Results
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. Moreover, because we had demonstrated the advantage of transfecting the tibialis cranialis muscle rather than the exposed quadriceps [11] this modification was also maintained.
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. electroporation was reported in 2008 for an interleukin-12 plasmid delivered into the tumors of patients suffering metastatic melanoma [7]. Close to 90 clinical trials involving electrotransfer have been performed subsequently and the technique is considered to be relatively inexpensive, flexible and safe because it does not introduce any additional chemicals or viruses into cells [8]. However, as far as we know, the utilization of naked DNA is still not at the clinical level for systemic diseases [5].
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. In previous work, the injection of the hGH gene into the tibialis cranialis (TA) muscle of 40 day old lit/scid mice provided a high catch-up growth of 76% for femur, 36% for nose-to-tail length and 39% for tail length [5]. Further studies on the different in vivo expressions of either hGH or mGH in these mice models could also take advantage of a recent review in which partially humanized transgenic mice were able to co-express hGH and mGH, offering a unique opportunity of investigation [12].
In the present study, we have carried out an initial optimization of this methodology to improve the transfection efficiency in the homologous model based on genomic mGH DNA electrotransfer into the tibialis muscle of immunocompetent lit/lit mice, taking into account the recommendation of the use of this model for setting up preclinical testing of non-viral gene therapy.

Results
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. Moreover, because we had demonstrated the advantage of transfecting the tibialis cranialis muscle rather than the exposed quadriceps [11] this modification was also maintained.
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.  was reported in 2008 for an interleukin-12 plasmid delivered into the tumors of patients suffering metastatic melanoma [7]. Close to 90 clinical trials involving electrotransfer have been performed subsequently and the technique is considered to be relatively inexpensive, flexible and safe because it does not introduce any additional chemicals or viruses into cells [8]. However, as far as we know, the utilization of naked DNA is still not at the clinical level for systemic diseases [5].
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. In previous work, the injection of the hGH gene into the tibialis cranialis (TA) muscle of 40 day old lit/scid mice provided a high catch-up growth of 76% for femur, 36% for nose-to-tail length and 39% for tail length [5]. Further studies on the different in vivo expressions of either hGH or mGH in these mice models could also take advantage of a recent review in which partially humanized transgenic mice were able to co-express hGH and mGH, offering a unique opportunity of investigation [12].
In the present study, we have carried out an initial optimization of this methodology to improve the transfection efficiency in the homologous model based on genomic mGH DNA electrotransfer into the tibialis muscle of immunocompetent lit/lit mice, taking into account the recommendation of the use of this model for setting up preclinical testing of non-viral gene therapy.

Results
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. Moreover, because we had demonstrated the advantage of transfecting the tibialis cranialis muscle rather than the exposed quadriceps [11] this modification was also maintained.
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. electroporation was reported in 2008 for an interleukin-12 plasmid delivered into the tumors of patients suffering metastatic melanoma [7]. Close to 90 clinical trials involving electrotransfer have been performed subsequently and the technique is considered to be relatively inexpensive, flexible and safe because it does not introduce any additional chemicals or viruses into cells [8]. However, as far as we know, the utilization of naked DNA is still not at the clinical level for systemic diseases [5].
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. In previous work, the injection of the hGH gene into the tibialis cranialis (TA) muscle of 40 day old lit/scid mice provided a high catch-up growth of 76% for femur, 36% for nose-to-tail length and 39% for tail length [5]. Further studies on the different in vivo expressions of either hGH or mGH in these mice models could also take advantage of a recent review in which partially humanized transgenic mice were able to co-express hGH and mGH, offering a unique opportunity of investigation [12].
In the present study, we have carried out an initial optimization of this methodology to improve the transfection efficiency in the homologous model based on genomic mGH DNA electrotransfer into the tibialis muscle of immunocompetent lit/lit mice, taking into account the recommendation of the use of this model for setting up preclinical testing of non-viral gene therapy.

Results
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. Moreover, because we had demonstrated the advantage of transfecting the tibialis cranialis muscle rather than the exposed quadriceps [11] this modification was also maintained.
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. In contrast to the lack of an improvement upon increasing the amount of DNA per injection, a stantial increase in expression efficiency was obtained upon increasing the number of injection s. This result is presented in Figure 2A, where 14.7 ± 3.7 ng/mL vs. 0.5 ± 0.2 ng/mL for saline (n = or each group; p < 0.001) were obtained when the approach of two injections into each tibialis nialis muscle was employed, in comparison with two injections into a single tibialis cranialis scle (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 ) negative control (saline); ( Molecules 2020, 25, x FOR PEER REVIEW comparison with the negative control: * p < 0. after a single administration into the right TA dissolved in 20 μL of saline, followed by th control (saline); 50 μg DNA; 75 μg D In contrast to the lack of an improvemen substantial increase in expression efficiency w sites. This result is presented in Figure 2A, wh 3 for each group; p < 0.001) were obtained w cranialis muscle was employed, in comparis muscle (5.9 ± 2.7 ng/mL, p < 0.02, n = 3). Even h ng/mL for the control group (n = 7 for each g In contrast to the lack of an improvement upon increasing the amount of DNA per injection, a ubstantial increase in expression efficiency was obtained upon increasing the number of injection ites. This result is presented in Figure 2A, where 14.7 ± 3.7 ng/mL vs. 0.5 ± 0.2 ng/mL for saline (n = for each group; p < 0.001) were obtained when the approach of two injections into each tibialis ranialis muscle was employed, in comparison with two injections into a single tibialis cranialis uscle (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 In contrast to the lack of an improvement upon increasing the amount of DNA per injection, a substantial increase in expression efficiency was obtained upon increasing the number of injection sites. 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 ) 100 µg DNA. (n = 3 animals per condition).
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.
In contrast to the lack of an improvement upon increasing the amount of DNA per injection, a substantial increase in expression efficiency was obtained upon increasing the number of injection sites. 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 ng/mL for the control group (n = 7 for each group; p < 0.001) were obtained in the serum of lit/scid mice three days after two injections of genomic hGH DNA plasmid into each tibialis cranialis muscle ( Figure 2B). In contrast to the lack of an improvement upon increasing the amount of DNA per injection, a substantial increase in expression efficiency was obtained upon increasing the number of injection sites. 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 ng/mL for the control group (n = 7 for each group; p < 0.001) were obtained in the serum of lit/scid mice three days after two injections of genomic hGH DNA plasmid into each tibialis cranialis muscle ( Figure 2B). negative control (saline); 2 injections into each TA muscle (n = 7 animals per condition). Significance test, in comparison with the negative control: * p < 0.05; ** p < 0.005; *** p < 0.001.
Based on these results a 36-day bioassay was carried out utilizing 60-day-old mice: 5 lit/lit mice as a saline-injected control; 7 lit/lit mice as the DNA-treated group and 5 lit/+ mice of the same strain and age as a positive control. Figure 3 shows the data for the variation of the weight of each group during the bioassay and the figure legend summarizes the best fit second-degree or quadratic polynomial fits of these data. The differences in the slopes of the three curves, as measured by the linear coefficient (B1) of each equation, are particularly evident and reflects the fact that the weight gain for the treated mice is significantly different (p < 0.0001) from that of the untreated negative control. The difference in slope between the curves for treated lit/lit and for heterozygous co-aged mice was non-significant. In contrast to the lack of an improvement upon increasing the amount of DNA per injection, a substantial increase in expression efficiency was obtained upon increasing the number of injection sites. 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 ng/mL for the control group (n = 7 for each group; p < 0.001) were obtained in the serum of lit/scid mice three days after two injections of genomic hGH DNA plasmid into each tibialis cranialis muscle ( Figure 2B). negative control (saline); 2 injections into each TA muscle (n = 7 animals per condition). Significance test, in comparison with the negative control: * p < 0.05; ** p < 0.005; *** p < 0.001.
Based on these results a 36-day bioassay was carried out utilizing 60-day-old mice: 5 lit/lit mice as a saline-injected control; 7 lit/lit mice as the DNA-treated group and 5 lit/+ mice of the same strain and age as a positive control. Figure 3 shows the data for the variation of the weight of each group during the bioassay and the figure legend summarizes the best fit second-degree or quadratic polynomial fits of these data. The differences in the slopes of the three curves, as measured by the linear coefficient (B1) of each equation, are particularly evident and reflects the fact that the weight gain for the treated mice is significantly different (p < 0.0001) from that of the untreated negative control. The difference in slope between the curves for treated lit/lit and for heterozygous co-aged mice was non-significant. In contrast to the lack of an improvement upon increasing the amount of DNA per injection, a substantial increase in expression efficiency was obtained upon increasing the number of injection sites. 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 ng/mL for the control group (n = 7 for each group; p < 0.001) were obtained in the serum of lit/scid mice three days after two injections of genomic hGH DNA plasmid into each tibialis cranialis muscle ( Figure 2B). negative control (saline); 2 injections into each TA muscle (n = 7 animals per condition). Significance test, in comparison with the negative control: * p < 0.05; ** p < 0.005; *** p < 0.001.
Based on these results a 36-day bioassay was carried out utilizing 60-day-old mice: 5 lit/lit mice as a saline-injected control; 7 lit/lit mice as the DNA-treated group and 5 lit/+ mice of the same strain and age as a positive control. Figure 3 shows the data for the variation of the weight of each group during the bioassay and the figure legend summarizes the best fit second-degree or quadratic polynomial fits of these data. The differences in the slopes of the three curves, as measured by the linear coefficient (B1) of each equation, are particularly evident and reflects the fact that the weight gain for the treated mice is significantly different (p < 0.0001) from that of the untreated negative control. The difference in slope between the curves for treated lit/lit and for heterozygous co-aged mice was non-significant. In contrast to the lack of an improvement upon increasing the amount of DNA per injection, a substantial increase in expression efficiency was obtained upon increasing the number of injection sites. 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 ng/mL for the control group (n = 7 for each group; p < 0.001) were obtained in the serum of lit/scid mice three days after two injections of genomic hGH DNA plasmid into each tibialis cranialis muscle ( Figure 2B). negative control (saline); 2 injections into each TA muscle (n = 7 animals per condition). Significance test, in comparison with the negative control: * p < 0.05; ** p < 0.005; *** p < 0.001.
Based on these results a 36-day bioassay was carried out utilizing 60-day-old mice: 5 lit/lit mice as a saline-injected control; 7 lit/lit mice as the DNA-treated group and 5 lit/+ mice of the same strain and age as a positive control. Figure 3 shows the data for the variation of the weight of each group during the bioassay and the figure legend summarizes the best fit second-degree or quadratic polynomial fits of these data. The differences in the slopes of the three curves, as measured by the linear coefficient (B1) of each equation, are particularly evident and reflects the fact that the weight gain for the treated mice is significantly different (p < 0.0001) from that of the untreated negative control. The difference in slope between the curves for treated lit/lit and for heterozygous co-aged mice was non-significant. In contrast to the lack of an improvement upon increasing the amount of DNA per injection, a substantial increase in expression efficiency was obtained upon increasing the number of injection sites. 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 ng/mL for the control group (n = 7 for each group; p < 0.001) were obtained in the serum of lit/scid mice three days after two injections of genomic hGH DNA plasmid into each tibialis cranialis muscle ( Figure 2B). negative control (saline); 2 injections into each TA muscle (n = 7 animals per condition). Significance test, in comparison with the negative control: * p < 0.05; ** p < 0.005; *** p < 0.001.
Based on these results a 36-day bioassay was carried out utilizing 60-day-old mice: 5 lit/lit mice as a saline-injected control; 7 lit/lit mice as the DNA-treated group and 5 lit/+ mice of the same strain and age as a positive control. Figure 3 shows the data for the variation of the weight of each group during the bioassay and the figure legend summarizes the best fit second-degree or quadratic polynomial fits of these data. The differences in the slopes of the three curves, as measured by the linear coefficient (B1) of each equation, are particularly evident and reflects the fact that the weight gain for the treated mice is significantly different (p < 0.0001) from that of the untreated negative control. The difference in slope between the curves for treated lit/lit and for heterozygous co-aged mice was non-significant. In contrast to the lack of an improvement upon increasing the amount of DNA per injection, a substantial increase in expression efficiency was obtained upon increasing the number of injection sites. 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 ng/mL for the control group (n = 7 for each group; p < 0.001) were obtained in the serum of lit/scid mice three days after two injections of genomic hGH DNA plasmid into each tibialis cranialis muscle ( Figure 2B). Negative control (saline); two injections into one TA muscle; 2 injections into each TA muscle (n = 3 animals per condition). (B). Serum hGH concentrations in lit/scid mice, 3 days after administration of 50 μg hGH plasmid DNA per injection (20 μL) in the TA muscle followed by electrotransfer based on the 375 V/cm protocol: negative control (saline); 2 injections into each TA muscle (n = 7 animals per condition). Significance test, in comparison with the negative control: * p < 0.05; ** p < 0.005; *** p < 0.001.
Based on these results a 36-day bioassay was carried out utilizing 60-day-old mice: 5 lit/lit mice as a saline-injected control; 7 lit/lit mice as the DNA-treated group and 5 lit/+ mice of the same strain and age as a positive control. Figure 3 shows the data for the variation of the weight of each group during the bioassay and the figure legend summarizes the best fit second-degree or quadratic polynomial fits of these data. The differences in the slopes of the three curves, as measured by the linear coefficient (B1) of each equation, are particularly evident and reflects the fact that the weight gain for the treated mice is significantly different (p < 0.0001) from that of the untreated negative control. The difference in slope between the curves for treated lit/lit and for heterozygous co-aged mice was non-significant. ) 2 injections into each TA muscle (n = 7 animals per condition). Significance test, in comparison with the negative control: * p < 0.05; ** p < 0.005; *** p < 0.001.
Based on these results a 36-day bioassay was carried out utilizing 60-day-old mice: 5 lit/lit mice as a saline-injected control; 7 lit/lit mice as the DNA-treated group and 5 lit/+ mice of the same strain and age as a positive control. Figure 3 shows the data for the variation of the weight of each group during the bioassay and the figure legend summarizes the best fit second-degree or quadratic polynomial fits of these data. The differences in the slopes of the three curves, as measured by the linear coefficient (B1) of each equation, are particularly evident and reflects the fact that the weight gain for the treated mice is significantly different (p < 0.0001) from that of the untreated negative control. The difference in slope between the curves for treated lit/lit and for heterozygous co-aged mice was non-significant. In addition to secretion efficiency, there was also a good sustainability of the mGH serum levels at the end of the 36-day assay. In DNA-treated mice, the average value of 8.5 ± 2.3 ng/mL (n = 7) was significantly higher (p < 0.02) than that presented by heterozygous mice (4.5 ± 1.5 ng/mL, n = 4) ( Figure  4). heterozygous mice, n = 4. Significance test, in comparison with the negative control: * p < 0.05; ** p < 0.001. Table 1 summarizes all of the important parameters obtained during the 36-day bioassay. Particularly noteworthy are the results for the catch-up growth, which range from 38 to 83%. These values are much higher than those obtained in previous work on lit/lit mice (14 to 24%) [10] and are comparable to the range of 36-76% for the catch-up growth obtained for lit/scid mice in a 60-day assay with 40-day old mice subjected to two injections per animal (on day 1 and on day 41) of hGH plasmid DNA [5]. In addition to secretion efficiency, there was also a good sustainability of the mGH serum levels at the end of the 36-day assay. In DNA-treated mice, the average value of 8.5 ± 2.3 ng/mL (n = 7) was significantly higher (p < 0.02) than that presented by heterozygous mice (4.5 ± 1.5 ng/mL, n = 4) ( Figure  4). heterozygous mice, n = 4. Significance test, in comparison with the negative control: * p < 0.05; ** p < 0.001. Table 1 summarizes all of the important parameters obtained during the 36-day bioassay. Particularly noteworthy are the results for the catch-up growth, which range from 38 to 83%. These values are much higher than those obtained in previous work on lit/lit mice (14 to 24%) [10] and are comparable to the range of 36-76% for the catch-up growth obtained for lit/scid mice in a 60-day assay with 40-day old mice subjected to two injections per animal (on day 1 and on day 41) of hGH plasmid DNA [5]. In addition to secretion efficiency, there was also a good sustainability of the mGH serum levels at the end of the 36-day assay. In DNA-treated mice, the average value of 8.5 ± 2.3 ng/mL (n = 7) was significantly higher (p < 0.02) than that presented by heterozygous mice (4.5 ± 1.5 ng/mL, n = 4) ( Figure  4). heterozygous mice, n = 4. Significance test, in comparison with the negative control: * p < 0.05; ** p < 0.001. Table 1 summarizes all of the important parameters obtained during the 36-day bioassay. Particularly noteworthy are the results for the catch-up growth, which range from 38 to 83%. These values are much higher than those obtained in previous work on lit/lit mice (14 to 24%) [10] and are comparable to the range of 36-76% for the catch-up growth obtained for lit/scid mice in a 60-day assay with 40-day old mice subjected to two injections per animal (on day 1 and on day 41) of hGH plasmid DNA [5]. In addition to secretion efficiency, there was also a good sustainability of the mGH serum levels at the end of the 36-day assay. In DNA-treated mice, the average value of 8.5 ± 2.3 ng/mL (n = 7) was significantly higher (p < 0.02) than that presented by heterozygous mice (4.5 ± 1.5 ng/mL, n = 4) ( Figure 4). In addition to secretion efficiency, there was also a good sustainability of the mGH serum levels at the end of the 36-day assay. In DNA-treated mice, the average value of 8.5 ± 2.3 ng/mL (n = 7) was significantly higher (p < 0.02) than that presented by heterozygous mice (4.5 ± 1.5 ng/mL, n = 4) ( Figure  4). heterozygous mice, n = 4. Significance test, in comparison with the negative control: * p < 0.05; ** p < 0.001. Table 1 summarizes all of the important parameters obtained during the 36-day bioassay. Particularly noteworthy are the results for the catch-up growth, which range from 38 to 83%. These values are much higher than those obtained in previous work on lit/lit mice (14 to 24%) [10] and are comparable to the range of 36-76% for the catch-up growth obtained for lit/scid mice in a 60-day assay with 40-day old mice subjected to two injections per animal (on day 1 and on day 41) of hGH plasmid DNA [5]. In addition to secretion efficiency, there was also a good sustainability of the mGH serum levels at the end of the 36-day assay. In DNA-treated mice, the average value of 8.5 ± 2.3 ng/mL (n = 7) was significantly higher (p < 0.02) than that presented by heterozygous mice (4.5 ± 1.5 ng/mL, n = 4) ( Figure  4). heterozygous mice, n = 4. Significance test, in comparison with the negative control: * p < 0.05; ** p < 0.001. Table 1 summarizes all of the important parameters obtained during the 36-day bioassay. Particularly noteworthy are the results for the catch-up growth, which range from 38 to 83%. These values are much higher than those obtained in previous work on lit/lit mice (14 to 24%) [10] and are comparable to the range of 36-76% for the catch-up growth obtained for lit/scid mice in a 60-day assay with 40-day old mice subjected to two injections per animal (on day 1 and on day 41) of hGH plasmid DNA [5]. In addition to secretion efficiency, there was also a good sustainability of the mGH serum levels at the end of the 36-day assay. In DNA-treated mice, the average value of 8.5 ± 2.3 ng/mL (n = 7) was significantly higher (p < 0.02) than that presented by heterozygous mice (4.5 ± 1.5 ng/mL, n = 4) ( Figure  4). heterozygous mice, n = 4. Significance test, in comparison with the negative control: * p < 0.05; ** p < 0.001. Table 1 summarizes all of the important parameters obtained during the 36-day bioassay. Particularly noteworthy are the results for the catch-up growth, which range from 38 to 83%. These values are much higher than those obtained in previous work on lit/lit mice (14 to 24%) [10] and are comparable to the range of 36-76% for the catch-up growth obtained for lit/scid mice in a 60-day assay with 40-day old mice subjected to two injections per animal (on day 1 and on day 41) of hGH plasmid DNA [5]. In addition to secretion efficiency, there was also a good sustainability of the mGH serum levels at the end of the 36-day assay. In DNA-treated mice, the average value of 8.5 ± 2.3 ng/mL (n = 7) was significantly higher (p < 0.02) than that presented by heterozygous mice (4.5 ± 1.5 ng/mL, n = 4) ( Figure  4). heterozygous mice, n = 4. Significance test, in comparison with the negative control: * p < 0.05; ** p < 0.001. Table 1 summarizes all of the important parameters obtained during the 36-day bioassay. Particularly noteworthy are the results for the catch-up growth, which range from 38 to 83%. These values are much higher than those obtained in previous work on lit/lit mice (14 to 24%) [10] and are comparable to the range of 36-76% for the catch-up growth obtained for lit/scid mice in a 60-day assay with 40-day old mice subjected to two injections per animal (on day 1 and on day 41) of hGH plasmid DNA [5].
) heterozygous mice, n = 4. Significance test, in comparison with the negative control: * p < 0.05; ** p < 0.001. Table 1 summarizes all of the important parameters obtained during the 36-day bioassay. Particularly noteworthy are the results for the catch-up growth, which range from 38 to 83%. These values are much higher than those obtained in previous work on lit/lit mice (14 to 24%) [10] and are comparable to the range of 36-76% for the catch-up growth obtained for lit/scid mice in a 60-day assay with 40-day old mice subjected to two injections per animal (on day 1 and on day 41) of hGH plasmid DNA [5]. Table 1. Growth parameters of lit/lit mice 36 days after administration of 50 µg of mGH plasmid DNA or saline into 2 sites of each TA muscle, followed by electroporation, in comparison with untreated heterozygous co-aged mice.