Comparison of wild-type and class I integrase mutant-FIV vectors in retina demonstrates sustained expression of integrated transgenes in retinal pigment epithelium.
BACKGROUND
In neonatal and adult rodent retina, substantial lentiviral vector expression has been detected primarily in retinal pigment epithelium (RPE), except in very young animals (2-5 days post-natal). In non-retinal tissues, studies of lentiviral vectors have utilized various controls. Among the most stringent are class I integrase mutants, which selectively block the integration reaction while leaving all other gag/pol-encoded functions intact. For HIV-1 vectors injected into brain, these have been used to simultaneously control for pseudotransduction and verify that long-term expression requires integration. Such experiments compare particles that differ only in a single amino acid within a single enzyme that forms a very small molar fraction of the virion. Class I integrase mutants have not been described for feline immunodeficiency virus (FIV) integrase, or tested in the eye for any lentiviral vector.


METHODS
We compared subretinally and intravitreally injected FIV vectors and followed animals for up to 7 months, a duration that exceeds prior studies. We also compared the wild-type (WT) vector with one incorporating a single class I amino acid mutation in FIV integrase (D66V). A mock vector (packaging construct absent) was an alternative control. All vectors were vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped and were injected on day 7 of life. One group of animals received either subretinal or intravitreal injections of WT vector in the right eyes. Control left eyes were injected with mock vector. These animals were sacrificed at 2 or 7 days post-injection. A second group received subretinal injections of either WT vector or equivalent D66V vector (reverse transcriptase-normalized to WT), and were analyzed after 2, 3 and 7 months. All eyes were scored for marker gene (beta-galactosidase) expression by an observer blinded to vector assignments.


RESULTS
Subretinal FIV vector injections were much more effective than intravitreal injections. The RPE was the principal retinal layer transduced by the WT vector, and at least 50% of the area of the retina expressed the marker gene at 3 and 7 months. Occasional cells in inner retinal layers also expressed beta-galactosidase at these time points. The sustained retinal expression produced by subretinally injected vector was blocked by the D66V mutation.


CONCLUSIONS
These results show that class I integrase mutant FIV vectors are useful control vectors, and that VSV-G-pseudotyped FIV vectors produce extensive retinal expression for at least 215 days, the longest duration yet reported for lentiviral vectors in retina. Transgene expression is mostly restricted to RPE after post-natal day 7 in rats, suggesting that FIV vectors could be used to target RPE for gene therapy.
Loewen
Nils
N
Molecular Medicine Program, Mayo Clinic, Rochester, MN 55905, USA.
Leske
David A
DA
Chen
Yi
Y
Teo
Wu-Lin
WL
Saenz
Dyana T
DT
Peretz
Mary
M
Holmes
Jonathan M
JM
Poeschla
Eric M
EM
D000367:Age Factors; D000818:Animals; D005786:Gene Expression Regulation; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D016089:Immunodeficiency Virus, Feline / Q000235:genetics* / Q000472:pathogenicity; D019426:Integrases / Q000235:genetics*; D009154:Mutation; D010857:Pigment Epithelium of Eye / Q000821:virology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000821:virology*; D019076:Transgenes
D019426:Integrases