Román Barraza

Unintegrated lentivirus DNA persistence and accessibility to expression in nondividing cells: analysis with class I integrase mutants.

ABSTRACT The circumstances under which unintegrated lentivirus DNA can persist and be a functional template for transcription and protein expression are not clear. We constructed and validated the first class I (nonpleiotropic) integrase (IN) mutants for a non-human lentivirus (feline immunodeficiency virus [FIV]) and analyzed both these and known class I human immunodeficiency virus type 1 IN mutants. The FIV IN mutants (D66V and D66V/D118A) had class I properties: Gag/Pol precursor expression, proteolytic processing, particle formation, and reverse transcriptase (RT) production were normal, while the transduction of dividing fibroblasts was prevented and integration was blocked. When injected into rat retinas, the wild-type (WT) vector produced extensive, persistent transgene expression, compared with only rare positive neuronal cells for the IN mutant vector. In contrast, both WT and mutant vectors produced entirely equivalent, effective transduction levels of primary rat neurons (retinal ganglion cells). By testing the hypothesis that the unexpected retinal neuron transduction was related to cell cycle status, we found that when fibroblasts were growth arrested, transduction and internally promoted transgene expression were not inhibited at all by the class I FIV or HIV-1 IN mutations. Cells were then transduced under aphidicolin arrest and were released from the block 48 h later. Vector expression was stable and durable during repeated passaging in WT vector-transduced cells, while the release of cells transduced with equivalent RT units of class I IN mutant FIV or HIV vector resulted in a steady decline of expression, from 97 to 0% of cells by day 10. Southern blot and PCR analyses showed a lack of integration, irrespective of cell cycle, for the class I mutants and an increase in one- and two-long terminal repeat circular and linear unintegrated DNAs in growth-arrested cells. We conclude that if cell division is prevented, unintegrated FIV and HIV-1 vector DNAs can produce high-level internally promoted transgene expression equivalent to WT vectors. The expression correlates with the unintegrated DNA levels. These observations may facilitate the study of the roles of IN and other preintegration complex components in preintegration phases of infection by (i) providing an alternative way to monitor unintegrated nuclear cDNA forms, (ii) restricting ascertainment to the transcriptionally functional subset of unintegrated DNA, (iii) enabling analysis in individual, nondividing cells, and (iv) uncoupling other potential functions of IN from integration.

Mapping the Encapsidation Determinants of Feline Immunodeficiency Virus

ABSTRACT Encapsidation of retroviral RNA involves specific interactions between viral proteins and cis-acting genomic RNA sequences. Human immunodeficiency virus type 1 (HIV-1) RNA encapsidation determinants appear to be more complex and dispersed than those of murine retroviruses. Feline lentiviral (feline immunodeficiency virus [FIV]) encapsidation has not been studied. To gain comparative insight into lentiviral encapsidation and to optimize FIV-based vectors, we used RNase protection assays of cellular and virion RNAs to determine packaging efficiencies of FIV deletion mutants, and we studied replicative phenotypes of mutant viruses. Unlike the case for other mammalian retroviruses, the sequences between the major splice donor (MSD) and the start codon of gag contribute negligibly to FIV encapsidation. Moreover, molecular clones having deletions in this region were replication competent. In contrast, sequences upstream of the MSD were important for encapsidation, and deletion of the U5 element markedly reduced genomic RNA packaging. The contribution of gag sequences to packaging was systematically investigated with subgenomic FIV vectors containing variable portions of the gag open reading frame, with all virion proteins supplied in trans. When no gag sequence was present, packaging was abolished and marker gene transduction was absent. Inclusion of the first 144 nucleotides (nt) of gag increased vector encapsidation to detectable levels, while inclusion of the first 311 nt increased it to nearly wild-type levels and resulted in high-titer FIV vectors. However, the identified proximal gag sequence is necessary but not sufficient, since viral mRNAs that contain all coding regions, with or without as much as 119 nt of adjacent upstream 5′ leader, were excluded from encapsidation. The results identify a mechanism whereby FIV can encapsidate its genomic mRNA in preference to subgenomic mRNAs.

Prostaglandin pathway gene therapy for sustained reduction of intraocular pressure.

Cyclooxygenase-2 (COX-2) is a rate-limiting enzyme in prostaglandin (PG) biosynthesis. In the eye, loss of COX-2 expression in aqueous humor-secreting cells has been associated with primary open-angle glaucoma (POAG). Reduction of intraocular pressure (IOP) is the main treatment goal in this disease. We used lentiviral vectors to stably express COX-2 and other PG biosynthesis and response transgenes in the ciliary body epithelium and trabecular meshwork (TM), the ocular suborgans that produce aqueous humor and regulate its outflow, respectively. We show that robust ectopic COX-2 expression and PG production require COX-2 complementary DNA (cDNA) sequence optimization. When COX-2 expression was coupled with a similarly optimized synthetic PGF2alpha receptor transgene to enable downstream signaling, gene therapy produced substantial and sustained reductions in IOP in a large animal model, the domestic cat. This study provides the first gene therapy for correcting the main cause of glaucoma.

Human gene therapy vectors derived from feline lentiviruses

Lentiviral vectors are useful for gene transfer to dividing and nondividing cells. Feline immunodeficiency virus (FIV) vectors transduce most human cell types with good efficiency and may have advantages for clinical gene therapy applications. This article reviews significant progress in the development and refinement of FIV vector systems.

Feline Immunodeficiency Virus-Based Lentiviral Vectors

Feline immunodeficiency virus (FIV)-based lentiviral vectors are useful for introducing integrated transgenes into nondividing human cells. This article describes the production and use of advanced generation FIV vectors. Key properties are discussed in comparison to other lentiviral vectors. Additional topics include the practical implications of species-specific retroviral restriction factors and the production of nonintegrating FIV vectors.

Surgical Correction of Childhood Intermittent Exotropia and the Risk of Developing Mental Illness

PURPOSE To assess whether successful surgical intervention for intermittent exotropia, or the timing of intervention, has any effect on the development of mental illness. DESIGN Retrospective, observational case series. METHODS All patients (<19 years of age) diagnosed with intermittent exotropia in Olmsted County, Minnesota, from January 1, 1975, through December 31, 1994, were reviewed retrospectively. Potential cases were identified using the resources of the Rochester Epidemiology Project, a medical records database designed to capture data on any patient-physician encounter in Olmsted County, Minnesota. The main outcome measures were the occurrence and severity of mental illness among those who underwent strabismus surgery compared with those who did not. RESULTS Ninety-six (52%) of the 184 children identified were diagnosed with a mental illness at a mean age of 23.3 years (range, 6 to 41 years). Thirty-five (36%) of the 96 children in whom mental illness developed underwent strabismus surgery. Success at surgery (<10 prism diopters) was not associated with a decreased occurrence of mental illness (P = .30). Of the 88 patients in whom mental illness did not develop, strabismus surgery was not more commonly performed (P = .54), nor was it performed at a younger age (P = 1.0), when compared with the 96 patients in whom mental illness developed later. CONCLUSIONS Strabismus surgery for children with intermittent exotropia, regardless of success or age at surgery, did not alter the development of mental illness by early adulthood.

Titration of Feline Immunodeficiency Virus-Based Lentiviral Vector Preparations

Feline immunodeficiency virus (FIV)-based lentiviral vectors are useful for introducing integrated transgenes into nondividing human cells. This protocol describes methods for measuring and calculating vector titers in transducing units (TU)/mL. Alternate methods are provided for green fluorescent protein (GFP) vectors and for β-galactosidase vectors.

Production and harvest of feline immunodeficiency virus-based lentiviral vector from cells grown in T75 tissue-culture flasks.

Feline immunodeficiency virus (FIV)-based lentiviral vectors are useful for introducing integrated transgenes into nondividing human cells. This protocol describes the production and harvesting of vector from cells grown in T75 tissue-culture flasks. The methods are for production for basic science laboratory use and in vivo experimentation. They do not meet standards for clinical-grade (good manufacturing practice [GMP]) production.