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Onset Time and Durability of Huntingtin Suppression in Rhesus Putamen After Direct Infusion of Antihuntingtin siRNA.

Grondin R, Ge P, Chen Q, Sutherland JE, Zhang Z, Gash DM, Stiles DK, Stewart GR, Sah DW, Kaemmerer WF - Mol Ther Nucleic Acids (2015)

Bottom Line: After a 1-week washout period, monkeys received radiolabeled siRNA targeting huntingtin.After 1 or 3 days of siRNA delivery, monkeys were either terminated, or their pumps were shut off and they were terminated 10 or 24 days later.These findings indicate the rapid onset and durability of siRNA-mediated target gene suppression observed in other organs also occurs in the brain, and support the use of episodic delivery of siRNA into the brain for treatment of Huntington's disease and possibly other neurodegenerative diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Neurobiology, University of Kentucky College of Medicine, Lexington, Kentucky, USA.

ABSTRACT
One possible treatment for Huntington's disease involves direct infusion of a small, interfering RNA (siRNA) designed to reduce huntingtin expression into brain tissue from a chronically implanted programmable pump. Here, we studied the suppression of huntingtin mRNA achievable with short infusion times, and investigated how long suppression may persist after infusion ceases. Rhesus monkeys received 3 days of infusion of Magnevist into the putamen to confirm catheter patency and fluid distribution. After a 1-week washout period, monkeys received radiolabeled siRNA targeting huntingtin. After 1 or 3 days of siRNA delivery, monkeys were either terminated, or their pumps were shut off and they were terminated 10 or 24 days later. Results indicate that the onset of huntingtin mRNA suppression in the rhesus putamen occurs rapidly, achieving a plateau throughout the putamen within 4 days. Conversely, loss of huntingtin suppression progresses slowly, persisting an estimated 27-39 days in the putamen and surrounding white matter. These findings indicate the rapid onset and durability of siRNA-mediated target gene suppression observed in other organs also occurs in the brain, and support the use of episodic delivery of siRNA into the brain for treatment of Huntington's disease and possibly other neurodegenerative diseases.

No MeSH data available.


Related in: MedlinePlus

Suppression durability in the putamen. (a) Mean and standard error of suppression averaged over all tissue punches from the putamen in monkeys terminated 0, 10 or 24 days after siRNA delivery cessation (groups 3, 4, and 5, respectively). Gray-filled bars are the suppression values based on the zero point provided by the common cDNA standard; white bars are the suppression values after subtracting the corresponding phosphate-buffered saline Group1 average from the same tisue punch locations. **P < 0.01 versus zero, *P < 0.05 (Tukey post-hoc test), †P < 0.10 versus zero, two-tailed; ns = not significant. (b) Linear, concave up, and concave down curve fit to the decline of suppression measured in RNA from punch location B14 from the putamen of monkeys at 0, 10, and 24 days after cessation of siRNA delivery using the suppression values based on the zero point provided by the common cDNA standard. Dotted line: best linear fit, Supp = 56.34 + (–1.21 * day); gray-dashed curve: best concave up fit, Supp = 56.13 * exp(–0.027 * day); solid black curve: best concave down fit, Supp = 54.10 – (exp(0.14 * day)). The curve fits for each punch location in the putamen are provided in the supplemental materials.
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fig6: Suppression durability in the putamen. (a) Mean and standard error of suppression averaged over all tissue punches from the putamen in monkeys terminated 0, 10 or 24 days after siRNA delivery cessation (groups 3, 4, and 5, respectively). Gray-filled bars are the suppression values based on the zero point provided by the common cDNA standard; white bars are the suppression values after subtracting the corresponding phosphate-buffered saline Group1 average from the same tisue punch locations. **P < 0.01 versus zero, *P < 0.05 (Tukey post-hoc test), †P < 0.10 versus zero, two-tailed; ns = not significant. (b) Linear, concave up, and concave down curve fit to the decline of suppression measured in RNA from punch location B14 from the putamen of monkeys at 0, 10, and 24 days after cessation of siRNA delivery using the suppression values based on the zero point provided by the common cDNA standard. Dotted line: best linear fit, Supp = 56.34 + (–1.21 * day); gray-dashed curve: best concave up fit, Supp = 56.13 * exp(–0.027 * day); solid black curve: best concave down fit, Supp = 54.10 – (exp(0.14 * day)). The curve fits for each punch location in the putamen are provided in the supplemental materials.

Mentions: Restricting the analysis to punches located within the putamen produced the following ANOVA results: there are significant differences among the experimental groups (F = 10.67, df = 2, 6, P = 0.010) and among the various punches (F = 2.29, df = 40, 203, P < 0.001). Furthermore, there is no evidence that the differences among experimental group varied by punch location (interaction of group × punch, F = 0.79, df = 80, 203, P = 0.891). That is, the time course of the suppression was similar regardless of the particular location of the punch within the putamen. Planned follow-up questions regarding the durability of suppression were addressed by T-tests at the various time points, separately, as shown in Figure 6a. Figure 6a also shows that when the percent suppression is normalized by subtracting the averages of the PBS group (Group 1), none of the other groups' averages are significantly different from zero, and the average of Group 5 (24 days after cessation of siRNA delivery) trends below zero (P = 0.079, two-tailed) which would be indicative of a “rebound” effect with even greater HTT expression (above baseline) after the siRNA treatment is stopped. However, there is no evidence of such a rebound effect in the HTT suppression values using zero point defined by the common cDNA standard; the apparent increase in HTT expression is an artifact of the mathematical subtraction of the PBS group values.


Onset Time and Durability of Huntingtin Suppression in Rhesus Putamen After Direct Infusion of Antihuntingtin siRNA.

Grondin R, Ge P, Chen Q, Sutherland JE, Zhang Z, Gash DM, Stiles DK, Stewart GR, Sah DW, Kaemmerer WF - Mol Ther Nucleic Acids (2015)

Suppression durability in the putamen. (a) Mean and standard error of suppression averaged over all tissue punches from the putamen in monkeys terminated 0, 10 or 24 days after siRNA delivery cessation (groups 3, 4, and 5, respectively). Gray-filled bars are the suppression values based on the zero point provided by the common cDNA standard; white bars are the suppression values after subtracting the corresponding phosphate-buffered saline Group1 average from the same tisue punch locations. **P < 0.01 versus zero, *P < 0.05 (Tukey post-hoc test), †P < 0.10 versus zero, two-tailed; ns = not significant. (b) Linear, concave up, and concave down curve fit to the decline of suppression measured in RNA from punch location B14 from the putamen of monkeys at 0, 10, and 24 days after cessation of siRNA delivery using the suppression values based on the zero point provided by the common cDNA standard. Dotted line: best linear fit, Supp = 56.34 + (–1.21 * day); gray-dashed curve: best concave up fit, Supp = 56.13 * exp(–0.027 * day); solid black curve: best concave down fit, Supp = 54.10 – (exp(0.14 * day)). The curve fits for each punch location in the putamen are provided in the supplemental materials.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4561652&req=5

fig6: Suppression durability in the putamen. (a) Mean and standard error of suppression averaged over all tissue punches from the putamen in monkeys terminated 0, 10 or 24 days after siRNA delivery cessation (groups 3, 4, and 5, respectively). Gray-filled bars are the suppression values based on the zero point provided by the common cDNA standard; white bars are the suppression values after subtracting the corresponding phosphate-buffered saline Group1 average from the same tisue punch locations. **P < 0.01 versus zero, *P < 0.05 (Tukey post-hoc test), †P < 0.10 versus zero, two-tailed; ns = not significant. (b) Linear, concave up, and concave down curve fit to the decline of suppression measured in RNA from punch location B14 from the putamen of monkeys at 0, 10, and 24 days after cessation of siRNA delivery using the suppression values based on the zero point provided by the common cDNA standard. Dotted line: best linear fit, Supp = 56.34 + (–1.21 * day); gray-dashed curve: best concave up fit, Supp = 56.13 * exp(–0.027 * day); solid black curve: best concave down fit, Supp = 54.10 – (exp(0.14 * day)). The curve fits for each punch location in the putamen are provided in the supplemental materials.
Mentions: Restricting the analysis to punches located within the putamen produced the following ANOVA results: there are significant differences among the experimental groups (F = 10.67, df = 2, 6, P = 0.010) and among the various punches (F = 2.29, df = 40, 203, P < 0.001). Furthermore, there is no evidence that the differences among experimental group varied by punch location (interaction of group × punch, F = 0.79, df = 80, 203, P = 0.891). That is, the time course of the suppression was similar regardless of the particular location of the punch within the putamen. Planned follow-up questions regarding the durability of suppression were addressed by T-tests at the various time points, separately, as shown in Figure 6a. Figure 6a also shows that when the percent suppression is normalized by subtracting the averages of the PBS group (Group 1), none of the other groups' averages are significantly different from zero, and the average of Group 5 (24 days after cessation of siRNA delivery) trends below zero (P = 0.079, two-tailed) which would be indicative of a “rebound” effect with even greater HTT expression (above baseline) after the siRNA treatment is stopped. However, there is no evidence of such a rebound effect in the HTT suppression values using zero point defined by the common cDNA standard; the apparent increase in HTT expression is an artifact of the mathematical subtraction of the PBS group values.

Bottom Line: After a 1-week washout period, monkeys received radiolabeled siRNA targeting huntingtin.After 1 or 3 days of siRNA delivery, monkeys were either terminated, or their pumps were shut off and they were terminated 10 or 24 days later.These findings indicate the rapid onset and durability of siRNA-mediated target gene suppression observed in other organs also occurs in the brain, and support the use of episodic delivery of siRNA into the brain for treatment of Huntington's disease and possibly other neurodegenerative diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Neurobiology, University of Kentucky College of Medicine, Lexington, Kentucky, USA.

ABSTRACT
One possible treatment for Huntington's disease involves direct infusion of a small, interfering RNA (siRNA) designed to reduce huntingtin expression into brain tissue from a chronically implanted programmable pump. Here, we studied the suppression of huntingtin mRNA achievable with short infusion times, and investigated how long suppression may persist after infusion ceases. Rhesus monkeys received 3 days of infusion of Magnevist into the putamen to confirm catheter patency and fluid distribution. After a 1-week washout period, monkeys received radiolabeled siRNA targeting huntingtin. After 1 or 3 days of siRNA delivery, monkeys were either terminated, or their pumps were shut off and they were terminated 10 or 24 days later. Results indicate that the onset of huntingtin mRNA suppression in the rhesus putamen occurs rapidly, achieving a plateau throughout the putamen within 4 days. Conversely, loss of huntingtin suppression progresses slowly, persisting an estimated 27-39 days in the putamen and surrounding white matter. These findings indicate the rapid onset and durability of siRNA-mediated target gene suppression observed in other organs also occurs in the brain, and support the use of episodic delivery of siRNA into the brain for treatment of Huntington's disease and possibly other neurodegenerative diseases.

No MeSH data available.


Related in: MedlinePlus