Bioluminescent imaging of a marking transgene and correction of Fabry mice by neonatal injection of recombinant lentiviral vectors

Abstract

Successful therapy for many inherited disorders could be improved if the intervention were initiated early. This is especially true for lysosomal storage disorders. Earlier intervention may allow metabolic correction to occur before lipid buildup has irreversible consequences and͞or before the immune system mounts limiting responses. We have been developing gene therapy to treat lysosomal storage disorders, especially Fabry disease. We describe studies directed toward metabolic correction in neonatal animals mediated by recombinant lentiviral vectors. To develop this method, we first injected a marking lentiviral vector that engineers expression of luciferase into the temporal vein of recipient neonatal animals. The use of a cooled charged-coupled device camera allowed us to track transgene expression over time in live animals. We observed intense luciferase expression in many tissues, including the brain, that did not diminish over 24 weeks. Next, we injected neonatal Fabry mice a single time with a therapeutic lentiviral vector engineered to express human ␣-galactosidase A. The injection procedure was well tolerated. We observed increased plasma levels of ␣-galactosidase A activity starting at our first plasma collection point (4 weeks). Levels of ␣-galactosidase A activity were found to be significantly elevated in many tissues even after 28 weeks. No immune response was observed against the corrective transgene product. Increased levels of enzyme activity also led to significant reduction of globotriaosylceramide in the liver, spleen, and heart. This approach provides a method to treat lysosomal storage disorders and other disorders before destructive manifestations occur.gene therapy ͉ lentivirus ͉ luciferase marking ͉ lysosomal storage disorder

Bystander killing of malignant cells via the delivery of engineered thymidine-active deoxycytidine kinase for suicide gene therapy of cancer

“…The LV vector encoding the control enhanced green fluorescent protein (eGFP) cDNA, pHR'-EF-eGFP-WPRE-SIN, was described previously. 32 Fidelity of all constructs was confirmed by sequencing. VSVg-pseudotyped LVs were generated by transient transfection of 293T cells (ATCC, Manassas, VA, Canada) with PEI (Sigma, Oakville, ON, Canada) using a three-plasmid system (pHR' plasmid construct, packaging plasmid pCMVDR8.91 packaging plasmid, and the pMD.G VSV-g expression plasmid).…”

“…31 The resulting dCK-IRES-huCD19D cassette was then subcloned downstream of the EF1-a promoter in our HIV1-based recombinant LV expression plasmid, pHR'-cPPT-EF-WPRE-SIN, described previously. 32 The resulting pHR'-EF-dCK.DM-WPRE-SIN vector was then modified by sitedirected mutagenesis (QuikChange II XL Site-Directed Mutagenesis Kit, Stratagene, La Jolla, CA) to generate the pHR'-EF-dCK.DM.S74E-WPRE-SIN vector encoding the R104M.D133A.S74E dCK triple-mutant (DM.S74E) cDNA. 24 The mutagenesis primers used were: S74E forward 5 0 -CAAGAT GAATTTGAGGAACTTACAATGGAGCAGAAAAATGGTGGGAATGTTC-3 0 and S74E reverse 5 0 -GAACATTCCCACCATTTTTCTGCTCCATTGTAAGTTCCTCA AATTCATCTTG-3 0 .…”

H2 relaxin overexpression increases in vivo prostate xenograft tumor growth and angiogenesis

“…20 Briefly, expression cassettes present in the pCMV-H2-IRES-eGFP and pCMV-IRESeGFP plasmids were amplified by PCR with primers containing ClaI and AscI restriction enzyme recognition sites at the 5 0 and 3 0…”

“…20 Briefly, expression cassettes present in the pCMV-H2-IRES-eGFP and pCMV-IRESeGFP plasmids were amplified by PCR with primers containing ClaI and AscI restriction enzyme recognition sites at the 5 0 and 3 0 ends, respectively. Forward primer was 5 0 -ccatcgattgattctgtggataaccg-3 0 and reverse primer was 5 0 -gtggcgcgcccggccgctttacttgtac-3 0 .…”

“…The cDNA fragments were resolved on a 1% agarose gel, extracted using the GeneClean II Kit (Qbiogene, Inc., Montreal, QC, Canada), digested with ClaI and AscI (New England Biolabs, Mississauga, ON, Canada) and ligated into the ClaI/AscI sites present in the modified pHR 0 lentiviral backbone. 20 Positive clones (pHR-cPPT-CMV-H2-IRES-eGFP-WPRE and pHR-cPPT-CMV-IRES-eGFP-WPRE) were identified by diagnostic restriction enzyme digestion analyses (Roche, Laval, QC, Canada) and subsequent DNA sequencing analysis (ACGT Corp., Toronto, ON, Canada). LV virions were produced by a transient triple-transfection method using 293T monolayers.…”