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Relative persistence of AAV serotype 1 vector genomes in dystrophic muscle.

Pacak CA, Conlon T, Mah CS, Byrne BJ - Genet Vaccines Ther (2008)

Bottom Line: Our data demonstrate prolonged vector genome persistence in skeletal muscle from the hindlimbs injected with the therapeutic transgene as compared to hindlimbs injected with the reporter gene.We observed loss of vector genomes in skeletal muscles that were there were not protected by the benefits of therapeutic gene transfer.In comparison, the therapeutic vector expressing sarcoglycan led to reduction or elimination of myofiber loss.

View Article: PubMed Central - HTML - PubMed

Affiliation: Powell Gene Therapy Center, University of Florida, Gainesville, FL, USA. christina.pacak@childrens.harvard.edu

ABSTRACT
The purpose of this study was to assess the behavior of pseudotyped recombinant adeno-associated virus type 1 (rAAV2/1) vector genomes in dystrophic skeletal muscle. A comparison was made between a therapeutic vector and a reporter vector by injecting the hindlimb in a mouse model of Limb Girdle Muscular Dystrophy Type 2D (LGMD-2D) prior to disease onset. We hypothesized that the therapeutic vector would establish long-term persistence through prevention of myofiber turnover. In contrast, the reporter vector genome copy number would diminish over time due to disease-associated muscle degradation. One day old alpha sarcoglycan knockout mice (sgca-/-) were injected with 1 x 10(11) vector genomes of rAAV2/1-tMCK-sgca in one hindlimb and the same dose of rAAV2/1-tMCK-LacZ in the contra lateral hindlimb. Newborn mice are tolerant of the foreign transgene allowing for long-term expression of both the marker and the therapeutic gene in the background. At 2 time-points following vector administration, hindlimb muscles were harvested and analyzed for LacZ or sarcoglycan expression. Our data demonstrate prolonged vector genome persistence in skeletal muscle from the hindlimbs injected with the therapeutic transgene as compared to hindlimbs injected with the reporter gene. We observed loss of vector genomes in skeletal muscles that were there were not protected by the benefits of therapeutic gene transfer. In comparison, the therapeutic vector expressing sarcoglycan led to reduction or elimination of myofiber loss. Mitigating the membrane instability inherent in dystrophic muscle was able to prolong the life of individual myofibers.

No MeSH data available.


Related in: MedlinePlus

Vector genome persistence. (A-C) Log graphs showing vector genome amounts in individual muscles of the lower sgca-/- mouse hindlimb at 4 or 12 months post administration of either rAAV2/1-tMCK-sgca (black bars) or rAAV2/1-tMCK-LacZ (grey bars). Greater persistence of vector genomes is observed in the sgca injected muscles (* indicates statistical significance [p-value ≤ 0.05], ** indicates p-value = 0.29). (A) Data for all muscles combined from the right (sgca injected) and the left (LacZ injected) hindlimbs at 4 or 12 months post injection. Muscles analyzed include: extensor digitorum longus (ED), gastrocnemius (Ga), soleus (So), tibialis anterior (TA), and quadriceps (Qu). (B) Combined (primarily) fast-twitch muscle data (ED and TA) at 4 and 12 months post injection. (C) Combined mixed/slow-twitch muscle data (Ga, So, and Qu) at 4 and 12 months post injection. (D) Bar graph depicting the differences in vector genome copy numbers in individual muscles at either 4 months (black) or 12 months (grey) post injection. Differences in expression levels between the two constructs were greater at 4 than at 12 months post administration. (E) Immunofluorescence image of a quadriceps muscle cryosection (2 months post rAAV2/1-tMCK-sgca administration) showing alpha-sarcoglycan located at the cell membrane (green) and nuclei maintained in the cell periphery (DAPI stain-blue). (F) β-galactosidase stained quadriceps muscle cryosection (2 months post rAAV2/1-tMCK-LacZ administration) showing staining in transduced myofibers (blue). (G-H) Images of extensor digitorum longus muscles (4 months post delivery of sgca or LacZ [respectively]). (I-J) Images of soleus muscles (4 months post delivery of sgca or LacZ [respectively]).
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Figure 1: Vector genome persistence. (A-C) Log graphs showing vector genome amounts in individual muscles of the lower sgca-/- mouse hindlimb at 4 or 12 months post administration of either rAAV2/1-tMCK-sgca (black bars) or rAAV2/1-tMCK-LacZ (grey bars). Greater persistence of vector genomes is observed in the sgca injected muscles (* indicates statistical significance [p-value ≤ 0.05], ** indicates p-value = 0.29). (A) Data for all muscles combined from the right (sgca injected) and the left (LacZ injected) hindlimbs at 4 or 12 months post injection. Muscles analyzed include: extensor digitorum longus (ED), gastrocnemius (Ga), soleus (So), tibialis anterior (TA), and quadriceps (Qu). (B) Combined (primarily) fast-twitch muscle data (ED and TA) at 4 and 12 months post injection. (C) Combined mixed/slow-twitch muscle data (Ga, So, and Qu) at 4 and 12 months post injection. (D) Bar graph depicting the differences in vector genome copy numbers in individual muscles at either 4 months (black) or 12 months (grey) post injection. Differences in expression levels between the two constructs were greater at 4 than at 12 months post administration. (E) Immunofluorescence image of a quadriceps muscle cryosection (2 months post rAAV2/1-tMCK-sgca administration) showing alpha-sarcoglycan located at the cell membrane (green) and nuclei maintained in the cell periphery (DAPI stain-blue). (F) β-galactosidase stained quadriceps muscle cryosection (2 months post rAAV2/1-tMCK-LacZ administration) showing staining in transduced myofibers (blue). (G-H) Images of extensor digitorum longus muscles (4 months post delivery of sgca or LacZ [respectively]). (I-J) Images of soleus muscles (4 months post delivery of sgca or LacZ [respectively]).

Mentions: Our results show that at both 4 and 12 months post virus administration there were a statistically significantly higher number of vector genomes present in those hindlimb muscles injected with rAAV2/1-tMCK-sgca than those injected with rAAV2/1-tMCK-LacZ (Figure 1A). At 2 months post administration differences in the number of transduced myofibers between each hindlimb (as demonstrated by immunohistochemistry and LacZ staining of frozen skeletal muscle cryosections to identify alpha sarcoglycan [green] and β-galactosidase [blue]) were subtle (Figure 1E–F) but increased over time (Figure 1G–J). Vector genome assessment of extensor digitorum longus (EDL), and the tibialis anterior (TA) were combined as they are both composed of predominately fast-twitch myofibers (Figure 1B). At 4 months post virus administration a significant difference in the ability of the two vectors to persist over time in dystrophic muscle became evident. This difference was still present but was not as profound at 12 months post injection since the total number of vector genomes in rAAV2/1-tMCK-sgca injected EDL, and TA muscles decreased over time.


Relative persistence of AAV serotype 1 vector genomes in dystrophic muscle.

Pacak CA, Conlon T, Mah CS, Byrne BJ - Genet Vaccines Ther (2008)

Vector genome persistence. (A-C) Log graphs showing vector genome amounts in individual muscles of the lower sgca-/- mouse hindlimb at 4 or 12 months post administration of either rAAV2/1-tMCK-sgca (black bars) or rAAV2/1-tMCK-LacZ (grey bars). Greater persistence of vector genomes is observed in the sgca injected muscles (* indicates statistical significance [p-value ≤ 0.05], ** indicates p-value = 0.29). (A) Data for all muscles combined from the right (sgca injected) and the left (LacZ injected) hindlimbs at 4 or 12 months post injection. Muscles analyzed include: extensor digitorum longus (ED), gastrocnemius (Ga), soleus (So), tibialis anterior (TA), and quadriceps (Qu). (B) Combined (primarily) fast-twitch muscle data (ED and TA) at 4 and 12 months post injection. (C) Combined mixed/slow-twitch muscle data (Ga, So, and Qu) at 4 and 12 months post injection. (D) Bar graph depicting the differences in vector genome copy numbers in individual muscles at either 4 months (black) or 12 months (grey) post injection. Differences in expression levels between the two constructs were greater at 4 than at 12 months post administration. (E) Immunofluorescence image of a quadriceps muscle cryosection (2 months post rAAV2/1-tMCK-sgca administration) showing alpha-sarcoglycan located at the cell membrane (green) and nuclei maintained in the cell periphery (DAPI stain-blue). (F) β-galactosidase stained quadriceps muscle cryosection (2 months post rAAV2/1-tMCK-LacZ administration) showing staining in transduced myofibers (blue). (G-H) Images of extensor digitorum longus muscles (4 months post delivery of sgca or LacZ [respectively]). (I-J) Images of soleus muscles (4 months post delivery of sgca or LacZ [respectively]).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2572159&req=5

Figure 1: Vector genome persistence. (A-C) Log graphs showing vector genome amounts in individual muscles of the lower sgca-/- mouse hindlimb at 4 or 12 months post administration of either rAAV2/1-tMCK-sgca (black bars) or rAAV2/1-tMCK-LacZ (grey bars). Greater persistence of vector genomes is observed in the sgca injected muscles (* indicates statistical significance [p-value ≤ 0.05], ** indicates p-value = 0.29). (A) Data for all muscles combined from the right (sgca injected) and the left (LacZ injected) hindlimbs at 4 or 12 months post injection. Muscles analyzed include: extensor digitorum longus (ED), gastrocnemius (Ga), soleus (So), tibialis anterior (TA), and quadriceps (Qu). (B) Combined (primarily) fast-twitch muscle data (ED and TA) at 4 and 12 months post injection. (C) Combined mixed/slow-twitch muscle data (Ga, So, and Qu) at 4 and 12 months post injection. (D) Bar graph depicting the differences in vector genome copy numbers in individual muscles at either 4 months (black) or 12 months (grey) post injection. Differences in expression levels between the two constructs were greater at 4 than at 12 months post administration. (E) Immunofluorescence image of a quadriceps muscle cryosection (2 months post rAAV2/1-tMCK-sgca administration) showing alpha-sarcoglycan located at the cell membrane (green) and nuclei maintained in the cell periphery (DAPI stain-blue). (F) β-galactosidase stained quadriceps muscle cryosection (2 months post rAAV2/1-tMCK-LacZ administration) showing staining in transduced myofibers (blue). (G-H) Images of extensor digitorum longus muscles (4 months post delivery of sgca or LacZ [respectively]). (I-J) Images of soleus muscles (4 months post delivery of sgca or LacZ [respectively]).
Mentions: Our results show that at both 4 and 12 months post virus administration there were a statistically significantly higher number of vector genomes present in those hindlimb muscles injected with rAAV2/1-tMCK-sgca than those injected with rAAV2/1-tMCK-LacZ (Figure 1A). At 2 months post administration differences in the number of transduced myofibers between each hindlimb (as demonstrated by immunohistochemistry and LacZ staining of frozen skeletal muscle cryosections to identify alpha sarcoglycan [green] and β-galactosidase [blue]) were subtle (Figure 1E–F) but increased over time (Figure 1G–J). Vector genome assessment of extensor digitorum longus (EDL), and the tibialis anterior (TA) were combined as they are both composed of predominately fast-twitch myofibers (Figure 1B). At 4 months post virus administration a significant difference in the ability of the two vectors to persist over time in dystrophic muscle became evident. This difference was still present but was not as profound at 12 months post injection since the total number of vector genomes in rAAV2/1-tMCK-sgca injected EDL, and TA muscles decreased over time.

Bottom Line: Our data demonstrate prolonged vector genome persistence in skeletal muscle from the hindlimbs injected with the therapeutic transgene as compared to hindlimbs injected with the reporter gene.We observed loss of vector genomes in skeletal muscles that were there were not protected by the benefits of therapeutic gene transfer.In comparison, the therapeutic vector expressing sarcoglycan led to reduction or elimination of myofiber loss.

View Article: PubMed Central - HTML - PubMed

Affiliation: Powell Gene Therapy Center, University of Florida, Gainesville, FL, USA. christina.pacak@childrens.harvard.edu

ABSTRACT
The purpose of this study was to assess the behavior of pseudotyped recombinant adeno-associated virus type 1 (rAAV2/1) vector genomes in dystrophic skeletal muscle. A comparison was made between a therapeutic vector and a reporter vector by injecting the hindlimb in a mouse model of Limb Girdle Muscular Dystrophy Type 2D (LGMD-2D) prior to disease onset. We hypothesized that the therapeutic vector would establish long-term persistence through prevention of myofiber turnover. In contrast, the reporter vector genome copy number would diminish over time due to disease-associated muscle degradation. One day old alpha sarcoglycan knockout mice (sgca-/-) were injected with 1 x 10(11) vector genomes of rAAV2/1-tMCK-sgca in one hindlimb and the same dose of rAAV2/1-tMCK-LacZ in the contra lateral hindlimb. Newborn mice are tolerant of the foreign transgene allowing for long-term expression of both the marker and the therapeutic gene in the background. At 2 time-points following vector administration, hindlimb muscles were harvested and analyzed for LacZ or sarcoglycan expression. Our data demonstrate prolonged vector genome persistence in skeletal muscle from the hindlimbs injected with the therapeutic transgene as compared to hindlimbs injected with the reporter gene. We observed loss of vector genomes in skeletal muscles that were there were not protected by the benefits of therapeutic gene transfer. In comparison, the therapeutic vector expressing sarcoglycan led to reduction or elimination of myofiber loss. Mitigating the membrane instability inherent in dystrophic muscle was able to prolong the life of individual myofibers.

No MeSH data available.


Related in: MedlinePlus