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

Translation of influence of injection volume to a large animal model, the rhesus macaque.RAG KO mice were injected with 1010 GC AAV8.CMV.201Ig IA either by IM or IV injection; tissues were harvested on day 56 and analyzed for vector genome copies (GC), quantified as GC/diploid genome in (A) liver and (B) muscle. (C) Biodistribution of AAV8 vector was determined on day 90 post-vector administration in a rhesus macaque. Vector was administered at a dose of 3×1012 GC/kg by IM injection into the vastus lateralis muscle of both right and left legs as 1 ml injections per kg body weight (vector concentration of 3×1012 GC/ml). DNA and RNA were extracted for quantification of GC (open bars) and transcript levels of 201Ig IA (closed bars), respectively. Values for muscle are the average of measurements at 12 sites throughout the injected muscle and liver is the average of the four lobes, which were quantified separately. There was no detectable GC or RNA in control (un-injected) muscle samples. LN, lymph node; Ax, axillary; In, inguinal; Mes, mesenteric; Il, iliac; Pop, popliteal. (D) Time course of expression of 201Ig IA in serum. (E) Rhesus macaques were injected IM with 3×1011 GC/kg of AAV8.CMV.201Ig IA, as either 1 ml vector injections per kg body weight (3×1011 GC/ml) or 0.1 ml injection per kg body weight (3×1012 GC/ml) (n = 2/group). Expression of 201Ig IA was measured in serum by ELISA and values are expressed as mean ± SEM. *p<0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112268-g006: Translation of influence of injection volume to a large animal model, the rhesus macaque.RAG KO mice were injected with 1010 GC AAV8.CMV.201Ig IA either by IM or IV injection; tissues were harvested on day 56 and analyzed for vector genome copies (GC), quantified as GC/diploid genome in (A) liver and (B) muscle. (C) Biodistribution of AAV8 vector was determined on day 90 post-vector administration in a rhesus macaque. Vector was administered at a dose of 3×1012 GC/kg by IM injection into the vastus lateralis muscle of both right and left legs as 1 ml injections per kg body weight (vector concentration of 3×1012 GC/ml). DNA and RNA were extracted for quantification of GC (open bars) and transcript levels of 201Ig IA (closed bars), respectively. Values for muscle are the average of measurements at 12 sites throughout the injected muscle and liver is the average of the four lobes, which were quantified separately. There was no detectable GC or RNA in control (un-injected) muscle samples. LN, lymph node; Ax, axillary; In, inguinal; Mes, mesenteric; Il, iliac; Pop, popliteal. (D) Time course of expression of 201Ig IA in serum. (E) Rhesus macaques were injected IM with 3×1011 GC/kg of AAV8.CMV.201Ig IA, as either 1 ml vector injections per kg body weight (3×1011 GC/ml) or 0.1 ml injection per kg body weight (3×1012 GC/ml) (n = 2/group). Expression of 201Ig IA was measured in serum by ELISA and values are expressed as mean ± SEM. *p<0.05.

Mentions: Mouse studies were performed with an AAV8 vector expressing 201Ig IA from a CMV promoter with tissues harvested at day 56. Animals were injected IM with 1010 GC of vector in different volumes (2×25 µl, 1×10 µl, and 1×2 µl). The deposition of vector significantly increased in liver as the concentration of the vector increased, consistent with the effects of vector concentration on transgene expression (Figure 6A). While there was not a significant increase in muscle, there was a trend towards higher GC following administration of smaller injection volumes (Figure 6B). The amount of vector present in the liver from the most concentrated dose delivered IM was close to, but slightly lower, than that observed following an IV injection of the same dose of vector (Figure 6A). Injection of vector IM in its most concentrated form yielded GC/diploid genome in muscle 2-fold lower than that in liver, again supporting substantial targeting of liver after IM injection of 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)

Translation of influence of injection volume to a large animal model, the rhesus macaque.RAG KO mice were injected with 1010 GC AAV8.CMV.201Ig IA either by IM or IV injection; tissues were harvested on day 56 and analyzed for vector genome copies (GC), quantified as GC/diploid genome in (A) liver and (B) muscle. (C) Biodistribution of AAV8 vector was determined on day 90 post-vector administration in a rhesus macaque. Vector was administered at a dose of 3×1012 GC/kg by IM injection into the vastus lateralis muscle of both right and left legs as 1 ml injections per kg body weight (vector concentration of 3×1012 GC/ml). DNA and RNA were extracted for quantification of GC (open bars) and transcript levels of 201Ig IA (closed bars), respectively. Values for muscle are the average of measurements at 12 sites throughout the injected muscle and liver is the average of the four lobes, which were quantified separately. There was no detectable GC or RNA in control (un-injected) muscle samples. LN, lymph node; Ax, axillary; In, inguinal; Mes, mesenteric; Il, iliac; Pop, popliteal. (D) Time course of expression of 201Ig IA in serum. (E) Rhesus macaques were injected IM with 3×1011 GC/kg of AAV8.CMV.201Ig IA, as either 1 ml vector injections per kg body weight (3×1011 GC/ml) or 0.1 ml injection per kg body weight (3×1012 GC/ml) (n = 2/group). Expression of 201Ig IA was measured in serum by ELISA and values are expressed as mean ± SEM. *p<0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112268-g006: Translation of influence of injection volume to a large animal model, the rhesus macaque.RAG KO mice were injected with 1010 GC AAV8.CMV.201Ig IA either by IM or IV injection; tissues were harvested on day 56 and analyzed for vector genome copies (GC), quantified as GC/diploid genome in (A) liver and (B) muscle. (C) Biodistribution of AAV8 vector was determined on day 90 post-vector administration in a rhesus macaque. Vector was administered at a dose of 3×1012 GC/kg by IM injection into the vastus lateralis muscle of both right and left legs as 1 ml injections per kg body weight (vector concentration of 3×1012 GC/ml). DNA and RNA were extracted for quantification of GC (open bars) and transcript levels of 201Ig IA (closed bars), respectively. Values for muscle are the average of measurements at 12 sites throughout the injected muscle and liver is the average of the four lobes, which were quantified separately. There was no detectable GC or RNA in control (un-injected) muscle samples. LN, lymph node; Ax, axillary; In, inguinal; Mes, mesenteric; Il, iliac; Pop, popliteal. (D) Time course of expression of 201Ig IA in serum. (E) Rhesus macaques were injected IM with 3×1011 GC/kg of AAV8.CMV.201Ig IA, as either 1 ml vector injections per kg body weight (3×1011 GC/ml) or 0.1 ml injection per kg body weight (3×1012 GC/ml) (n = 2/group). Expression of 201Ig IA was measured in serum by ELISA and values are expressed as mean ± SEM. *p<0.05.
Mentions: Mouse studies were performed with an AAV8 vector expressing 201Ig IA from a CMV promoter with tissues harvested at day 56. Animals were injected IM with 1010 GC of vector in different volumes (2×25 µl, 1×10 µl, and 1×2 µl). The deposition of vector significantly increased in liver as the concentration of the vector increased, consistent with the effects of vector concentration on transgene expression (Figure 6A). While there was not a significant increase in muscle, there was a trend towards higher GC following administration of smaller injection volumes (Figure 6B). The amount of vector present in the liver from the most concentrated dose delivered IM was close to, but slightly lower, than that observed following an IV injection of the same dose of vector (Figure 6A). Injection of vector IM in its most concentrated form yielded GC/diploid genome in muscle 2-fold lower than that in liver, again supporting substantial targeting of liver after IM injection of 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