Limits...
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.

Show MeSH

Related in: MedlinePlus

Liver expression following IM vector administration in mice.Visualization of differential ffLuc expression patterns using Xenogen whole-body bioluminescent imaging on day 7 post-IM administration of 1010 GC AAV8.CMV.ffLuc (A) or AAV8.TBG.ffLuc (B) to C57BL/6 mice. Vector was administered as one 10 µl injection into the gastrocnemius muscle of the right leg. (C) ffLuc expression was quantified at day 28 post-vector administration by ffLuc tissue assays. ffLuc expression measured as RLU normalized to the total protein concentration of the organ. Data are presented as fold change over background where background was RLU/total protein of organ (g) in control tissues from un-injected mice. (D) Comparison of expression of 201Ig IA from the CMV and TBG promoters following a 10 µl IM injection in RAG KO mice. Expression of 201Ig IA in serum was measured by ELISA. Values are expressed as mean ± SEM (n = 4/group). ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4230988&req=5

pone-0112268-g001: Liver expression following IM vector administration in mice.Visualization of differential ffLuc expression patterns using Xenogen whole-body bioluminescent imaging on day 7 post-IM administration of 1010 GC AAV8.CMV.ffLuc (A) or AAV8.TBG.ffLuc (B) to C57BL/6 mice. Vector was administered as one 10 µl injection into the gastrocnemius muscle of the right leg. (C) ffLuc expression was quantified at day 28 post-vector administration by ffLuc tissue assays. ffLuc expression measured as RLU normalized to the total protein concentration of the organ. Data are presented as fold change over background where background was RLU/total protein of organ (g) in control tissues from un-injected mice. (D) Comparison of expression of 201Ig IA from the CMV and TBG promoters following a 10 µl IM injection in RAG KO mice. Expression of 201Ig IA in serum was measured by ELISA. Values are expressed as mean ± SEM (n = 4/group). ***p<0.001.

Mentions: AAV8 vector expressing firefly luciferase (ffLuc) from the ubiquitous CMV promoter was injected IM into C57BL/6 mice at a dose of 1010 genome copies (GC) per mouse (Figure 1A). Vector was administered as one 10 µl injection into the right gastrocnemius muscle and on day 7 post-vector mice were imaged to determine the localization of ffLuc expression. Significant ffLuc expression, which localized to both the injected muscle and the liver, was demonstrated (Figure 1A). These imaging results were quantitatively reproduced and further expanded using biochemical assays where ffLuc was measured in both liver and muscle samples and normalized to total protein on day 28 post-vector administration (Figure 1C). IM injection of vector using the CMV promoter for expression resulted in extensive ffLuc expression in the injected muscle with concordant expression in the liver, which was 321-fold over background (control un-injected mice).


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)

Liver expression following IM vector administration in mice.Visualization of differential ffLuc expression patterns using Xenogen whole-body bioluminescent imaging on day 7 post-IM administration of 1010 GC AAV8.CMV.ffLuc (A) or AAV8.TBG.ffLuc (B) to C57BL/6 mice. Vector was administered as one 10 µl injection into the gastrocnemius muscle of the right leg. (C) ffLuc expression was quantified at day 28 post-vector administration by ffLuc tissue assays. ffLuc expression measured as RLU normalized to the total protein concentration of the organ. Data are presented as fold change over background where background was RLU/total protein of organ (g) in control tissues from un-injected mice. (D) Comparison of expression of 201Ig IA from the CMV and TBG promoters following a 10 µl IM injection in RAG KO mice. Expression of 201Ig IA in serum was measured by ELISA. Values are expressed as mean ± SEM (n = 4/group). ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112268-g001: Liver expression following IM vector administration in mice.Visualization of differential ffLuc expression patterns using Xenogen whole-body bioluminescent imaging on day 7 post-IM administration of 1010 GC AAV8.CMV.ffLuc (A) or AAV8.TBG.ffLuc (B) to C57BL/6 mice. Vector was administered as one 10 µl injection into the gastrocnemius muscle of the right leg. (C) ffLuc expression was quantified at day 28 post-vector administration by ffLuc tissue assays. ffLuc expression measured as RLU normalized to the total protein concentration of the organ. Data are presented as fold change over background where background was RLU/total protein of organ (g) in control tissues from un-injected mice. (D) Comparison of expression of 201Ig IA from the CMV and TBG promoters following a 10 µl IM injection in RAG KO mice. Expression of 201Ig IA in serum was measured by ELISA. Values are expressed as mean ± SEM (n = 4/group). ***p<0.001.
Mentions: AAV8 vector expressing firefly luciferase (ffLuc) from the ubiquitous CMV promoter was injected IM into C57BL/6 mice at a dose of 1010 genome copies (GC) per mouse (Figure 1A). Vector was administered as one 10 µl injection into the right gastrocnemius muscle and on day 7 post-vector mice were imaged to determine the localization of ffLuc expression. Significant ffLuc expression, which localized to both the injected muscle and the liver, was demonstrated (Figure 1A). These imaging results were quantitatively reproduced and further expanded using biochemical assays where ffLuc was measured in both liver and muscle samples and normalized to total protein on day 28 post-vector administration (Figure 1C). IM injection of vector using the CMV promoter for expression resulted in extensive ffLuc expression in the injected muscle with concordant expression in the liver, which was 321-fold over background (control un-injected mice).

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