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Intramuscular injection of AAV8 in mice and macaques is associated with substantial hepatic targeting and transgene expression.

Greig JA, Peng H, Ohlstein J, Medina-Jaszek CA, Ahonkhai O, Mentzinger A, Grant RL, Roy S, Chen SJ, Bell P, Tretiakova AP, Wilson JM - PLoS ONE (2014)

Bottom Line: We found that following IM administration of AAV8 vectors in mice, a portion of the vector reached the liver and hepatic gene expression contributed significantly to total expression of secreted transgenes.Hepatic distribution of vector following IM injection was also noted in rhesus macaques.These pre-clinical data on AAV delivery should inform safe and efficient development of future AAV products.

View Article: PubMed Central - PubMed

Affiliation: Gene Therapy Program, Department of Pathology and Laboratory Medicine, Division of Transfusion Medicine, University of Pennsylvania, TRL Suite 2000, 125 South 31st Street, Philadelphia, PA, 19104, United States of America.

ABSTRACT
Intramuscular (IM) administration of adeno-associated viral (AAV) vectors has entered the early stages of clinical development with some success, including the first approved gene therapy product in the West called Glybera. In preparation for broader clinical development of IM AAV vector gene therapy, we conducted detailed pre-clinical studies in mice and macaques evaluating aspects of delivery that could affect performance. We found that following IM administration of AAV8 vectors in mice, a portion of the vector reached the liver and hepatic gene expression contributed significantly to total expression of secreted transgenes. The contribution from liver could be controlled by altering injection volume and by the use of traditional (promoter) and non-traditional (tissue-specific microRNA target sites) expression control elements. Hepatic distribution of vector following IM injection was also noted in rhesus macaques. These pre-clinical data on AAV delivery should inform safe and efficient development of future AAV products.

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Reduction in IM injection volume increases transgene expression.1010 GC AAV8 expressing ffLuc from the CMV or TBG promoter was delivered IM as either two 25 µl injections (one into each leg), one 10 µl injection or one 2 µl injection to C57BL/6 mice. IV injections were performed as a 100 µl injection via the tail vein. ffLuc tissue assays were performed on tissue harvested at day 28 and normalized to the total protein concentration of liver (A) and muscle (B). RAG KO mice were administered with 1010 GC AAV8.CMV.201Ig IA (C) or AAV8.TBG.201Ig IA (D) by IV or IM injections, performed as described previously. Data for the one 10 µl injection groups were previously presented in Figure 1. Expression of 201Ig IA in serum was measured by ELISA. Values are expressed as mean ± SEM (n = 4/group). *p<0.05, **p<0.01, ***p<0.001.
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pone-0112268-g003: Reduction in IM injection volume increases transgene expression.1010 GC AAV8 expressing ffLuc from the CMV or TBG promoter was delivered IM as either two 25 µl injections (one into each leg), one 10 µl injection or one 2 µl injection to C57BL/6 mice. IV injections were performed as a 100 µl injection via the tail vein. ffLuc tissue assays were performed on tissue harvested at day 28 and normalized to the total protein concentration of liver (A) and muscle (B). RAG KO mice were administered with 1010 GC AAV8.CMV.201Ig IA (C) or AAV8.TBG.201Ig IA (D) by IV or IM injections, performed as described previously. Data for the one 10 µl injection groups were previously presented in Figure 1. Expression of 201Ig IA in serum was measured by ELISA. Values are expressed as mean ± SEM (n = 4/group). *p<0.05, **p<0.01, ***p<0.001.

Mentions: We investigated the impact of injection volume on the level and distribution of transgene expression. In these studies the same dose of vector was injected in a range of volumes from two 25 µl injections, one into each leg for a total injection volume of 50 µl, to one 2 µl injection in one site representing a 25-fold range of vector concentrations. The initial studies focused on mice injected with 1010 GC AAV8 vectors expressing ffLuc from the CMV or TBG promoters. Tissues were harvested at day 28 and transgene expression measured in lysates from liver (Figure 3A) and muscle (Figure 3B). Our original hypothesis was that increasing the volume for a fixed dose would increase the relative distribution of vector to liver. These studies did confirm the pilot experiments described in Figure 1, which used a single concentration of vector, whereby: 1) IM injection of vector resulted in substantial levels of transgene expression in liver, and 2) vectors using the TBG promoter produced levels of transgene product in liver following IM injection that were almost equivalent to the levels achieved when the same vector was injected IV. However, we were surprised to see that the more concentrated IM injections of vector did not help restrict expression to muscle; in fact these studies showed higher levels of liver ffLuc expression with lower volumes of vector. Also, the higher concentrations of vector yielded higher levels of overall ffLuc in both muscle and liver, independent of promoter. Based on in vitro experiments (data not shown), there was no significant loss of vector following dilution to different concentrations prior to administration in mice. Therefore, all mice received the same dose of vector and differences in distribution of the vector were due to the concentration of the injected vector.


Intramuscular injection of AAV8 in mice and macaques is associated with substantial hepatic targeting and transgene expression.

Greig JA, Peng H, Ohlstein J, Medina-Jaszek CA, Ahonkhai O, Mentzinger A, Grant RL, Roy S, Chen SJ, Bell P, Tretiakova AP, Wilson JM - PLoS ONE (2014)

Reduction in IM injection volume increases transgene expression.1010 GC AAV8 expressing ffLuc from the CMV or TBG promoter was delivered IM as either two 25 µl injections (one into each leg), one 10 µl injection or one 2 µl injection to C57BL/6 mice. IV injections were performed as a 100 µl injection via the tail vein. ffLuc tissue assays were performed on tissue harvested at day 28 and normalized to the total protein concentration of liver (A) and muscle (B). RAG KO mice were administered with 1010 GC AAV8.CMV.201Ig IA (C) or AAV8.TBG.201Ig IA (D) by IV or IM injections, performed as described previously. Data for the one 10 µl injection groups were previously presented in Figure 1. Expression of 201Ig IA in serum was measured by ELISA. Values are expressed as mean ± SEM (n = 4/group). *p<0.05, **p<0.01, ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4230988&req=5

pone-0112268-g003: Reduction in IM injection volume increases transgene expression.1010 GC AAV8 expressing ffLuc from the CMV or TBG promoter was delivered IM as either two 25 µl injections (one into each leg), one 10 µl injection or one 2 µl injection to C57BL/6 mice. IV injections were performed as a 100 µl injection via the tail vein. ffLuc tissue assays were performed on tissue harvested at day 28 and normalized to the total protein concentration of liver (A) and muscle (B). RAG KO mice were administered with 1010 GC AAV8.CMV.201Ig IA (C) or AAV8.TBG.201Ig IA (D) by IV or IM injections, performed as described previously. Data for the one 10 µl injection groups were previously presented in Figure 1. Expression of 201Ig IA in serum was measured by ELISA. Values are expressed as mean ± SEM (n = 4/group). *p<0.05, **p<0.01, ***p<0.001.
Mentions: We investigated the impact of injection volume on the level and distribution of transgene expression. In these studies the same dose of vector was injected in a range of volumes from two 25 µl injections, one into each leg for a total injection volume of 50 µl, to one 2 µl injection in one site representing a 25-fold range of vector concentrations. The initial studies focused on mice injected with 1010 GC AAV8 vectors expressing ffLuc from the CMV or TBG promoters. Tissues were harvested at day 28 and transgene expression measured in lysates from liver (Figure 3A) and muscle (Figure 3B). Our original hypothesis was that increasing the volume for a fixed dose would increase the relative distribution of vector to liver. These studies did confirm the pilot experiments described in Figure 1, which used a single concentration of vector, whereby: 1) IM injection of vector resulted in substantial levels of transgene expression in liver, and 2) vectors using the TBG promoter produced levels of transgene product in liver following IM injection that were almost equivalent to the levels achieved when the same vector was injected IV. However, we were surprised to see that the more concentrated IM injections of vector did not help restrict expression to muscle; in fact these studies showed higher levels of liver ffLuc expression with lower volumes of vector. Also, the higher concentrations of vector yielded higher levels of overall ffLuc in both muscle and liver, independent of promoter. Based on in vitro experiments (data not shown), there was no significant loss of vector following dilution to different concentrations prior to administration in mice. Therefore, all mice received the same dose of vector and differences in distribution of the vector were due to the concentration of the injected vector.

Bottom Line: We found that following IM administration of AAV8 vectors in mice, a portion of the vector reached the liver and hepatic gene expression contributed significantly to total expression of secreted transgenes.Hepatic distribution of vector following IM injection was also noted in rhesus macaques.These pre-clinical data on AAV delivery should inform safe and efficient development of future AAV products.

View Article: PubMed Central - PubMed

Affiliation: Gene Therapy Program, Department of Pathology and Laboratory Medicine, Division of Transfusion Medicine, University of Pennsylvania, TRL Suite 2000, 125 South 31st Street, Philadelphia, PA, 19104, United States of America.

ABSTRACT
Intramuscular (IM) administration of adeno-associated viral (AAV) vectors has entered the early stages of clinical development with some success, including the first approved gene therapy product in the West called Glybera. In preparation for broader clinical development of IM AAV vector gene therapy, we conducted detailed pre-clinical studies in mice and macaques evaluating aspects of delivery that could affect performance. We found that following IM administration of AAV8 vectors in mice, a portion of the vector reached the liver and hepatic gene expression contributed significantly to total expression of secreted transgenes. The contribution from liver could be controlled by altering injection volume and by the use of traditional (promoter) and non-traditional (tissue-specific microRNA target sites) expression control elements. Hepatic distribution of vector following IM injection was also noted in rhesus macaques. These pre-clinical data on AAV delivery should inform safe and efficient development of future AAV products.

Show MeSH
Related in: MedlinePlus