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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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Localization of LacZ expression in injected muscle following IM injection of AAV8 in C57BL/6 mice.Localization of LacZ expression in injected muscle was determined at day 21 post-vector administration in C57BL/6 mice. Vector was administered at a dose of 1010 GC as two 25 µl injections into the right and left legs (A, B) or as one 2 µl injection (C, D). A lower dose of 109 GC of vector was administered as two 25 µl injections into the right and left legs (E, F) or as one 2 µl injection (G, H). Three sections were taken throughout the injected gastrocnemius muscle of one representative animal per group (A, C, E and G) with one representative liver section per group (B, D, F and H) (n = 4/group).
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pone-0112268-g004: Localization of LacZ expression in injected muscle following IM injection of AAV8 in C57BL/6 mice.Localization of LacZ expression in injected muscle was determined at day 21 post-vector administration in C57BL/6 mice. Vector was administered at a dose of 1010 GC as two 25 µl injections into the right and left legs (A, B) or as one 2 µl injection (C, D). A lower dose of 109 GC of vector was administered as two 25 µl injections into the right and left legs (E, F) or as one 2 µl injection (G, H). Three sections were taken throughout the injected gastrocnemius muscle of one representative animal per group (A, C, E and G) with one representative liver section per group (B, D, F and H) (n = 4/group).

Mentions: A series of studies were performed using LacZ as a reporter gene to evaluate distribution of transduction at a cellular level as a function of vector concentration and dose. C57BL/6 mice were injected IM with AAV8 expressing LacZ from the CMV promoter. Liver and muscle tissue were harvested for analysis by LacZ histochemical staining 21 days post-vector administration (Figure 4). Sections of the gastrocnemius muscles were taken at intervals throughout the entire injected muscle. At a dose of 1010 GC, IM injection of vector as either two 25 µl injections (Figure 4A) or one 2 µl injection (Figure 4C) produced similar patterns of expression throughout the injected muscle, with a few transduced cells being seen in the liver (Figures 4B, 4D); note that the CMV promoter does not express well in liver. To determine if gene expression was saturated in the muscle at a dose of 1010 GC per mouse, the dose was lowered to 109 GC per mouse and a very different transduction pattern was seen for the two injection volumes (Figures 4E, 4G). Injection of the vector IM as two 25 µl injections produced few transduced cells in the muscle (Figure 4E). When the concentration of the vector was increased to enable injection of the same dose of vector in a volume of 2 µl, a large area of transduced skeletal muscle cells were seen (Figure 4G). This transduced area extended through multiple muscle sections. Again, only a few transduced cells were seen in the liver at a vector dose of 109 GC per mouse (Figures 4F, 4H). Therefore, administration of the same vector dose as a smaller injection volume improved skeletal muscle transduction at lower overall vector doses.


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)

Localization of LacZ expression in injected muscle following IM injection of AAV8 in C57BL/6 mice.Localization of LacZ expression in injected muscle was determined at day 21 post-vector administration in C57BL/6 mice. Vector was administered at a dose of 1010 GC as two 25 µl injections into the right and left legs (A, B) or as one 2 µl injection (C, D). A lower dose of 109 GC of vector was administered as two 25 µl injections into the right and left legs (E, F) or as one 2 µl injection (G, H). Three sections were taken throughout the injected gastrocnemius muscle of one representative animal per group (A, C, E and G) with one representative liver section per group (B, D, F and H) (n = 4/group).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4230988&req=5

pone-0112268-g004: Localization of LacZ expression in injected muscle following IM injection of AAV8 in C57BL/6 mice.Localization of LacZ expression in injected muscle was determined at day 21 post-vector administration in C57BL/6 mice. Vector was administered at a dose of 1010 GC as two 25 µl injections into the right and left legs (A, B) or as one 2 µl injection (C, D). A lower dose of 109 GC of vector was administered as two 25 µl injections into the right and left legs (E, F) or as one 2 µl injection (G, H). Three sections were taken throughout the injected gastrocnemius muscle of one representative animal per group (A, C, E and G) with one representative liver section per group (B, D, F and H) (n = 4/group).
Mentions: A series of studies were performed using LacZ as a reporter gene to evaluate distribution of transduction at a cellular level as a function of vector concentration and dose. C57BL/6 mice were injected IM with AAV8 expressing LacZ from the CMV promoter. Liver and muscle tissue were harvested for analysis by LacZ histochemical staining 21 days post-vector administration (Figure 4). Sections of the gastrocnemius muscles were taken at intervals throughout the entire injected muscle. At a dose of 1010 GC, IM injection of vector as either two 25 µl injections (Figure 4A) or one 2 µl injection (Figure 4C) produced similar patterns of expression throughout the injected muscle, with a few transduced cells being seen in the liver (Figures 4B, 4D); note that the CMV promoter does not express well in liver. To determine if gene expression was saturated in the muscle at a dose of 1010 GC per mouse, the dose was lowered to 109 GC per mouse and a very different transduction pattern was seen for the two injection volumes (Figures 4E, 4G). Injection of the vector IM as two 25 µl injections produced few transduced cells in the muscle (Figure 4E). When the concentration of the vector was increased to enable injection of the same dose of vector in a volume of 2 µl, a large area of transduced skeletal muscle cells were seen (Figure 4G). This transduced area extended through multiple muscle sections. Again, only a few transduced cells were seen in the liver at a vector dose of 109 GC per mouse (Figures 4F, 4H). Therefore, administration of the same vector dose as a smaller injection volume improved skeletal muscle transduction at lower overall vector doses.

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