Systemic correction of skeletal muscles in GAA-KO mice required IV administration of a very high number of AAV vector particles.14
In order to increase transduction of hindlimb muscles with fewer vector particles, an AAV vector containing the highly active muscle-specific MHCK7 regulatory cassette to drive human placental alkaline phosphatase (AP) expression was administered by ILP.20
AP staining demonstrated widespread transduced myofibers in the quadriceps, gastrocnimus and extensor digitorum longus (EDL) muscle following ILP administration. IV administration of the equivalent number of vector particles resulted in much less transduction of skeletal muscles and much more transduction of cardiac muscle, in comparison with ILP administration ().
Transduction of myofibers following hydrostatic ILP or IV administration of an AAV2/8 vector
We next confirmed that isolated limb perfusion (ILP) of the AAV2/8 vector greatly increased AAV transduction and GAA expression in multiple hindlimb muscles of GAA-KO mice. AAV-MHCK7hGAApA, which contains the MHCK7 regulatory cassette to drive human GAA expression, was packaged as AAV2/8 and administered to 3-month-old GAA-KO mice by hydrostatic ILP or IV injection, and control mice were administered PBS by ILP injection. Eighteen weeks following vector administration, high level of GAA expression were detected in multiple limb muscles including the gastrocnemius, soleus, EDL and quadriceps, in ILP vector-injected mice (). The efficiency of ILP administration was higher in three out of 6 GAA-KO with lower glycogen content in the gastrocnemius, and the ILP results were sorted into two groups accordingly (; ILP, High vs Low; p=0.049). Biochemical correction was confirmed by demonstrating that glycogen reduction was marked following ILP in the hindlimb muscles, including gastrocnemius (64%), soleus (70%), EDL (38%), and quadriceps (43%), in comparison with PBS-injected GAA-KO mice.
Biochemical evaluation of striated muscle transduction with an AAV2/8 vector delivered by hydrostatic ILP
In contrast, the IV-injected mice were found to have elevated GAA activity only in the heart, not in skeletal muscles (). Glycogen content was not reduced in the hindlimb muscles following IV administration, in comparison with PBS-injected GAA-KO mice (). Furthermore, glycogen content was reduced to a greater extent following ILP in comparison with IV administration in quadriceps (p=0.02), soleus (p=0.01), and EDL (p=0.03) (). The mean glycogen content of gastrocnemius for the higher efficiency ILP group (ILP High) was significantly reduced in comparison with the IV group (p=0.008); however, combining the results for all ILP-treated mice did not reveal significantly reduced glycogen content in the gastrocnemius in comparison with the IV group (p=0.06). The differing response between ILP High and Low groups reflected variations in transduction efficiency with hydrostatic ILP over the course of the initial experiment, which did not preclude significantly improved biochemical correction in 75% of the skeletal muscles analyzed over that achieved by IV administration. Differences in anti-GAA antibody levels were detected by ELISA between groups of GAA-KO mice following ILP and IV injections. The antibody levels were higher in ILP injected mice when compared with IV injected mice, and did not interfere with efficacy in terms of biochemical correction of striated muscle (). Furthermore, efficacy in skeletal muscle did correlate with vector genome quantification. For gastrocnemius, vector genomes were increased following ILP administration, in comparison with IV administration (0.11±0.05 versus 0.001±0.001, respectively), as quantified by Realtime PCR.
The pattern of GAA expression was further confirmed by Western blot analysis of muscles from each group following AAV2/8 vector administration (). Glycogen staining revealed glycogen clearance in most myofibers in the gastrocneminus of the ILP vector-injected mouse (). Despite the presence of anti-GAA antibodies in all vector-injected mice (not shown), long-term GAA expression and glycogen clearance was achieved in AAV-transduced muscle cells.
Western blot detection of GAA expression in striated muscle of GAA-KO mice with an AAV2/8 vector
Glycogen staining of skeletal muscle
Subsequently the number of vector particles was further reduced to evaluate the efficacy of ILP at a lower dose. Previously AAV2/9 was deemed a more efficient pseudotype for the correction of skeletal muscle than AAV2/8 in GAA-KO mice.14
The AAV2/9 vector was administered at a lower dose (3×1010
vp), either by ILP or IV administration.
Immune responses that might reduce efficacy were prevented by treating the tolerant GAA-KO mouse strain. GAA-KO mice were previously rendered immune tolerant to human GAA through the insertion of a low-expressing liver-specific transgene that prevented antibody formation in response to long-term ERT 5
, Immune tolerant GAA-KO mice would be anticipated to achieve a higher degree of efficacy from a given dose of AAV vector, due to the potential for secretion of GAA from transduced myofibers and the receptor-mediated uptake of GAA by nontransduced myofibers. Initially the absence of antibody formation was confirmed by persistent expression of GAA in plasma in vector treated mice as detected by Western blot (not shown). Consistent with earlier results, ILP administration significantly increased the GAA activity of hindlimb muscles other than quadriceps (p<0.05), in comparison with IV administration (). All mice treated with ILP responded demonstrated similarly reduced glycogen content in the hindlimb muscle during the second experiment, possibly related to increased experience with the method. Glycogen accumulations were significantly reduced in the gastrocnemius (p=0.0005), soleus (p=0.0004), and EDL (p=0.03) muscles by ILP administration, in comparison with IV (). However, IV administration significantly elevated GAA activity (p=3 ×10−8
) and reduced glycogen content in the heart (p=0.0007) in comparison with ILP administration (), confirming the advantage of systemic delivery with regard to correction of cardiac muscle.
Biochemical correction following ILP administration of an AAV2/9 vector