Recombinant adeno-associated virus type 2 pseudotypes: comparing safety, specificity, and transduction efficiency in the primate striatum. Laboratory investigation.
OBJECTIVE
Although several clinical trials utilizing the adeno-associated virus (AAV) type 2 serotype 2 (2/2) are now underway, it is unclear whether this particular serotype offers any advantage over others in terms of safety or efficiency when delivered directly to the CNS.


METHODS
Recombinant AAV2-green fluorescent protein (GFP) serotypes 2/1, 2/2, 2/5, and 2/8 were generated following standard triple transfection protocols (final yield 5.4 × 10(12) particles/ml). A total of 180 μl of each solution was stereotactically infused, covering the entire rostrocaudal extent of the caudoputamen in 4 rhesus monkeys (Macaca mulatta) (3.0 ± 0.5 kg). After 6 weeks' survival, the brain was formalin fixed, cut at 40 μm, and stained with standard immunohistochemistry for anti-GFP, anticaspase-2, and cell-specific markers (anti-microtubule-associated protein-2 for neurons and anti-glial fibrillary acidic protein for glia). Unbiased stereological counting methods were used to determine cell number and striatal volume.


RESULTS
The entire striatum of each animal contained GFP-positive cells with significant labeling extending beyond the borders of the basal ganglia. No ischemic/necrotic, hemorrhagic, or neoplastic change was observed in any brain. Total infusate volumes were similar across the 4 serotypes. However, GFP-labeled cell density was markedly different. Adeno-associated virus 2/1, 2/2, and 2/5 each labeled < 8000 cells/mm(3), whereas serotype 8 labeled > 21,000 cells, a 3- to 4-fold higher transduction efficiency. On the other hand, serotype 8 also labeled neurons and glia with equal affinity compared with neuronal specificities > 89% for the other serotypes. Moderate caspase-2 colabeling was noted in neurons immediately around the AAV2/1 injection tracts, but was not seen above the background anywhere in the brain following injections with serotypes 2, 5, or 8.


CONCLUSIONS
Intrastriatal delivery of AAV2 yields the highest cell transduction efficiencies but lowest neuronal specificity for serotype 8 when compared with serotypes 1, 2, and 5. Only AAV2/1 revealed significant caspase-2 activation. Careful consideration of serotype-specific differences in AAV2 neurotropism, transduction efficiency, and potential toxicity may affect future human trials.
Department of Neurosurgery, Massachusetts General Hospital, Boston, USA.
Carlos E
CE
Sanchez
Travis S
TS
Tierney
John T
JT
Gale
Kambiz N
KN
Alavian
Ayguen
A
Sahin
Jeng-Shin
JS
Lee
Richard C
RC
Mulligan
Bob S
BS
Carter
D000818:Animals; D053148:Caspase 3 / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics*; D018014:Gene Transfer Techniques / Q000009:adverse effects; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D007150:Immunohistochemistry; D008253:Macaca mulatta; D008279:Magnetic Resonance Imaging; D017072:Neostriatum / Q000166:cytology / Q000502:physiology*; D012449:Safety; D013238:Stereotaxic Techniques; D014161:Transduction, Genetic
D049452:Green Fluorescent Proteins; D053148:Caspase 3