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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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Effect of microRNA target sites on localization and expression level following IM injection of AAV8 in mice.Differential ffLuc expression patterns were visualized on day 7 post-IM administration of 1010 GC AAV8.CMV.ffLuc (A), AAV8.CMV.ffLuc.miR-122 (B) or AAV8.CMV.ffLuc.miR-206 (C). ffLuc expressing vectors were administered to C57BL/6 mice in two 10 µl injections into the right and left gastrocnemius muscles. (D) Liver and muscle expression of ffLuc was quantified separately on day 28 post-vector administration. Following substitution of ffLuc for 201Ig IA, expression of 201Ig IA was determined in RAG KO mice. Mice were injected with 1011 GC of vectors, administered as described previously and expression of 201Ig IA in serum was measured by ELISA (E). GC of the 201Ig IA vectors present in the liver of RAG KO mice at day 90 post-vector administration was determined (F). Values are expressed as mean ± SEM (n = 3/group). *p<0.05, ***p<0.001.
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pone-0112268-g005: Effect of microRNA target sites on localization and expression level following IM injection of AAV8 in mice.Differential ffLuc expression patterns were visualized on day 7 post-IM administration of 1010 GC AAV8.CMV.ffLuc (A), AAV8.CMV.ffLuc.miR-122 (B) or AAV8.CMV.ffLuc.miR-206 (C). ffLuc expressing vectors were administered to C57BL/6 mice in two 10 µl injections into the right and left gastrocnemius muscles. (D) Liver and muscle expression of ffLuc was quantified separately on day 28 post-vector administration. Following substitution of ffLuc for 201Ig IA, expression of 201Ig IA was determined in RAG KO mice. Mice were injected with 1011 GC of vectors, administered as described previously and expression of 201Ig IA in serum was measured by ELISA (E). GC of the 201Ig IA vectors present in the liver of RAG KO mice at day 90 post-vector administration was determined (F). Values are expressed as mean ± SEM (n = 3/group). *p<0.05, ***p<0.001.

Mentions: A critical issue not addressed in the experiments described above is the relative contribution of liver versus muscle in the production of secreted transgene products following IM injection of AAV8 vectors. Additional studies focused on vectors expressing the antibody 201Ig IA from a CMV promoter to simulate the likely clinical application of AAV expressed antibodies for prevention of infections, such as HIV and influenza [9], [10], [36], [37]. The strategy employed to address this issue utilized microRNA target sites that allow for tissue specific ablation of transgene expression. For example, the microRNA target sites for the liver-specific and the skeletal muscle-specific microRNAs, miR-122 and 206, respectively, have been previously shown to reduce gene expression in liver and muscle following the incorporation of these target sites within an AAV vector genome [32], [33], [34], [35]. Initial studies were performed with AAV8 vectors expressing ffLuc from a CMV promoter, with and without miR-122 and miR-206 target sites, in order to confirm the activity of the microRNA target sites. C57BL/6 mice injected IM with the ffLuc vectors were imaged for ffLuc expression on day 7 (Figures 5A-C) and expression was quantified on day 28 (Figure 5D). Incorporation of target sites for miR-122 and miR-206 into the 3′ UTR of the transgene produced specific knockdown of gene expression in the liver and muscle of 6.6- and 112-fold, respectively (Figures 5A-D). As previously demonstrated [32], [33], [35], [44], [45], [46], expression of the transgene in the organ that was not the target of microRNA-induced knockdown of gene expression remained unchanged (Figures 5A-D). These studies confirmed the usefulness of these microRNA target sites in specifically ablating liver or muscle expression.


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)

Effect of microRNA target sites on localization and expression level following IM injection of AAV8 in mice.Differential ffLuc expression patterns were visualized on day 7 post-IM administration of 1010 GC AAV8.CMV.ffLuc (A), AAV8.CMV.ffLuc.miR-122 (B) or AAV8.CMV.ffLuc.miR-206 (C). ffLuc expressing vectors were administered to C57BL/6 mice in two 10 µl injections into the right and left gastrocnemius muscles. (D) Liver and muscle expression of ffLuc was quantified separately on day 28 post-vector administration. Following substitution of ffLuc for 201Ig IA, expression of 201Ig IA was determined in RAG KO mice. Mice were injected with 1011 GC of vectors, administered as described previously and expression of 201Ig IA in serum was measured by ELISA (E). GC of the 201Ig IA vectors present in the liver of RAG KO mice at day 90 post-vector administration was determined (F). Values are expressed as mean ± SEM (n = 3/group). *p<0.05, ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112268-g005: Effect of microRNA target sites on localization and expression level following IM injection of AAV8 in mice.Differential ffLuc expression patterns were visualized on day 7 post-IM administration of 1010 GC AAV8.CMV.ffLuc (A), AAV8.CMV.ffLuc.miR-122 (B) or AAV8.CMV.ffLuc.miR-206 (C). ffLuc expressing vectors were administered to C57BL/6 mice in two 10 µl injections into the right and left gastrocnemius muscles. (D) Liver and muscle expression of ffLuc was quantified separately on day 28 post-vector administration. Following substitution of ffLuc for 201Ig IA, expression of 201Ig IA was determined in RAG KO mice. Mice were injected with 1011 GC of vectors, administered as described previously and expression of 201Ig IA in serum was measured by ELISA (E). GC of the 201Ig IA vectors present in the liver of RAG KO mice at day 90 post-vector administration was determined (F). Values are expressed as mean ± SEM (n = 3/group). *p<0.05, ***p<0.001.
Mentions: A critical issue not addressed in the experiments described above is the relative contribution of liver versus muscle in the production of secreted transgene products following IM injection of AAV8 vectors. Additional studies focused on vectors expressing the antibody 201Ig IA from a CMV promoter to simulate the likely clinical application of AAV expressed antibodies for prevention of infections, such as HIV and influenza [9], [10], [36], [37]. The strategy employed to address this issue utilized microRNA target sites that allow for tissue specific ablation of transgene expression. For example, the microRNA target sites for the liver-specific and the skeletal muscle-specific microRNAs, miR-122 and 206, respectively, have been previously shown to reduce gene expression in liver and muscle following the incorporation of these target sites within an AAV vector genome [32], [33], [34], [35]. Initial studies were performed with AAV8 vectors expressing ffLuc from a CMV promoter, with and without miR-122 and miR-206 target sites, in order to confirm the activity of the microRNA target sites. C57BL/6 mice injected IM with the ffLuc vectors were imaged for ffLuc expression on day 7 (Figures 5A-C) and expression was quantified on day 28 (Figure 5D). Incorporation of target sites for miR-122 and miR-206 into the 3′ UTR of the transgene produced specific knockdown of gene expression in the liver and muscle of 6.6- and 112-fold, respectively (Figures 5A-D). As previously demonstrated [32], [33], [35], [44], [45], [46], expression of the transgene in the organ that was not the target of microRNA-induced knockdown of gene expression remained unchanged (Figures 5A-D). These studies confirmed the usefulness of these microRNA target sites in specifically ablating liver or muscle expression.

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