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1.
Gene Ther ; 23(6): 548-56, 2016 06.
Article in English | MEDLINE | ID: mdl-27052802

ABSTRACT

Adeno-associated virus (AAV) vector-based gene therapy is a promising treatment strategy for delivery of neurotrophic transgenes to retinal ganglion cells (RGCs) in glaucoma patients. Retinal distribution of transgene expression following intravitreal injection (IVT) of AAV is variable in animal models and the vitreous humor may represent a barrier to initial vector penetration. The primary goal of our study was to investigate the effect of prior core vitrectomy with posterior hyaloid membrane peeling on pattern and efficiency of transduction of a capsid amino acid substituted AAV2 vector, carrying the green fluorescent protein (GFP) reporter transgene following IVT in dogs. When progressive intraocular inflammation developed starting 4 weeks post IVT, the study plan was modified to allow detailed characterization of the etiology as a secondary goal. Unexpectedly, surgical vitrectomy was found to significantly limit transduction, whereas in non-vitrectomized eyes transduction efficiency reached upwards to 37.3% of RGC layer cells. The developing retinitis was characterized by mononuclear cell infiltrates resulting from a delayed-type hypersensitivity reaction, which we suspect was directed at the GFP transgene. Our results, in a canine large animal model, support caution when considering surgical vitrectomy before IVT for retinal gene therapy in patients, as prior vitrectomy appears to significantly reduce transduction efficiency and may predispose the patient to development of vector-induced immune reactions.


Subject(s)
Dependovirus/genetics , Vitrectomy , Animals , Dogs , Genetic Vectors , Green Fluorescent Proteins/genetics , Humans , Retina/metabolism , Transduction, Genetic , Transgenes
3.
Gene Ther ; 23(2): 223-30, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26467396

ABSTRACT

Delivery of therapeutic transgenes to retinal photoreceptors using adeno-associated virus (AAV) vectors has traditionally required subretinal injection. Recently, photoreceptor transduction efficiency following intravitreal injection (IVT) has improved in rodent models through use of capsid-mutant AAV vectors; but remains limited in large animal models. Thickness of the inner limiting membrane (ILM) in large animals is thought to impair retinal penetration by AAV. Our study compared two newly developed AAV vectors containing multiple capsid amino acid substitutions following IVT in dogs. The ability of two promoter constructs to restrict reporter transgene expression to photoreceptors was also evaluated. AAV vectors containing the interphotoreceptor-binding protein (IRBP) promoter drove expression exclusively in rod and cone photoreceptors, with transduction efficiencies of ~4% of cones and 2% of rods. Notably, in the central region containing the cone-rich visual streak, 15.6% of cones were transduced. Significant regional variation existed, with lower transduction efficiencies in the temporal regions of all eyes. This variation did not correlate with ILM thickness. Vectors carrying a cone-specific promoter failed to transduce a quantifiable percentage of cone photoreceptors. The newly developed AAV vectors containing the IRBP promoter were capable of producing photoreceptor-specific transgene expression following IVT in the dog.


Subject(s)
Dependovirus/genetics , Gene Transfer Techniques , Genetic Vectors , Photoreceptor Cells, Vertebrate/metabolism , Animals , Dogs , Enhancer Elements, Genetic , Eye Proteins/genetics , Eye Proteins/metabolism , Genetic Vectors/genetics , Genetic Vectors/metabolism , Green Fluorescent Proteins/genetics , Intravitreal Injections , Promoter Regions, Genetic , Retina/metabolism , Transduction, Genetic
4.
Gene Ther ; 21(10): 913-20, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25056608

ABSTRACT

The cat is emerging as a promising large animal model for preclinical testing of retinal dystrophy therapies, for example, by gene therapy. However, there is a paucity of studies investigating viral vector gene transfer to the feline retina. We therefore sought to study the tropism of recombinant adeno-associated viral (rAAV) vectors for the feline outer retina. We delivered four rAAV serotypes: rAAV2/2, rAAV2/5, rAAV2/8 and rAAV2/9, each expressing green fluorescent protein (GFP) under the control of a cytomegalovirus promoter, to the subretinal space in cats and, for comparison, mice. Cats were monitored for gene expression by in vivo imaging and cellular tropism was determined using immunohistochemistry. In cats, rAAV2/2, rAAV2/8 and rAAV2/9 vectors induced faster and stronger GFP expression than rAAV2/5 and all vectors transduced the retinal pigment epithelium (RPE) and photoreceptors. Unlike in mice, cone photoreceptors in the cat retina were more efficiently transduced than rod photoreceptors. In mice, rAAV2/2 only transduced the RPE whereas the other vectors also transduced rods and cones. These results highlight species differences in cellular tropism of rAAV vectors in the outer retina. We conclude that rAAV serotypes are suitable for use for retinal gene therapy in feline models, particularly when cone photoreceptors are the target cell.


Subject(s)
Dependovirus/physiology , Green Fluorescent Proteins/metabolism , Retinal Cone Photoreceptor Cells/metabolism , Retinal Rod Photoreceptor Cells/metabolism , Animals , Cats , Dependovirus/genetics , Female , Genetic Therapy , Genetic Vectors/administration & dosage , Green Fluorescent Proteins/genetics , Injections, Intraocular , Male , Mice , Retinal Cone Photoreceptor Cells/virology , Retinal Rod Photoreceptor Cells/virology , Transduction, Genetic , Viral Tropism
5.
Gene Ther ; 21(1): 96-105, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24225638

ABSTRACT

Recombinant adeno-associated viruses are important vectors for retinal gene delivery. Currently utilized vectors have relatively slow onset, and for efficient transduction it is necessary to deliver treatment subretinally, with the potential for damage to the retina. Amino-acid substitutions in the viral capsid improve efficiency in rodent eyes by evading host responses. As dogs are important large animal models for human retinitis pigmentosa, we evaluated the speed and efficiency of retinal transduction using capsid-mutant vectors injected both subretinally and intravitreally. We evaluated AAV serotypes 2 and 8 with amino-acid substitutions of surface-exposed capsid tyrosine residues. The chicken beta-actin promoter was used to drive green fluorescent protein expression. Twelve normal adult beagles were injected; four dogs received intravitreal injections and eight dogs received subretinal injections. Capsid-mutant viruses tested included AAV2(quad Y-F) (intravitreal and subretinal) and self-complementary scAAV8(Y733F) (subretinal only). Contralateral control eyes received injections of scAAV5 (subretinal) or scAAV2 (intravitreal). Subretinally delivered vectors had a faster expression onset than intravitreally delivered vectors. Subretinally delivered scAAV8(Y733F) had a faster onset of expression than scAAV5. All subretinally injected vector types transduced the outer retina with high efficiency and the inner retina with moderate efficiency. Intravitreally delivered AAV2(quad Y-F) had a marginally higher efficiency of transduction of both outer retinal and inner retinal cells than scAAV2. Because of their rapid expression onset and efficient transduction, subretinally delivered capsid-mutant AAV8 vectors may increase the efficacy of gene therapy treatment for rapid photoreceptor degenerative diseases. With further refinement, capsid-mutant AAV2 vectors show promise for retinal gene delivery from an intravitreal approach.


Subject(s)
Capsid , Dependovirus/genetics , Genetic Vectors , Retina/metabolism , Amino Acid Substitution , Animals , Dependovirus/physiology , Dogs , Female , Humans , Injections, Intraocular , Male , Mutation , Recombinant Proteins/metabolism , Retina/virology , Transduction, Genetic , Tyrosine , Viral Tropism
6.
Gene Ther ; 20(5): 545-55, 2013 May.
Article in English | MEDLINE | ID: mdl-22951453

ABSTRACT

Recent clinical trials of retinal pigment epithelium gene (RPE65) supplementation therapy in Leber congenital amaurosis type 2 patients have demonstrated improvements in rod and cone function, but it may be some years before the effects of therapy on photoreceptor survival become apparent. The Rpe65-deficient dog is a very useful pre-clinical model in which to test efficacy of therapies, because the dog has a retina with a high degree of similarity to that of humans. In this study, we evaluated the effect of RPE65 gene therapy on photoreceptor survival in order to predict the potential benefit and limitations of therapy in patients. We examined the retinas of Rpe65-deficient dogs after RPE65 gene therapy to evaluate the preservation of rods and cone photoreceptor subtypes. We found that gene therapy preserves both rods and cones. While the moderate loss of rods in the Rpe65-deficient dog retina is slowed by gene therapy, S-cones are lost extensively and gene therapy can prevent that loss, although only within the treated area. Although LM-cones are not lost extensively, cone opsin mislocalization indicates that they are stressed, and this can be partially reversed by gene therapy. Our results suggest that gene therapy may be able to slow cone degeneration in patients if intervention is sufficiently early and also that it is probably important to treat the macula in order to preserve central function.


Subject(s)
Leber Congenital Amaurosis/therapy , Retinal Cone Photoreceptor Cells , Retinal Rod Photoreceptor Cells , cis-trans-Isomerases/genetics , Animals , Cell Survival/genetics , Disease Models, Animal , Dogs , Genetic Therapy , Leber Congenital Amaurosis/genetics , Leber Congenital Amaurosis/pathology , Retina/drug effects , Retina/pathology , Retinal Cone Photoreceptor Cells/drug effects , Retinal Cone Photoreceptor Cells/pathology , Retinal Rod Photoreceptor Cells/drug effects , Retinal Rod Photoreceptor Cells/pathology , cis-trans-Isomerases/administration & dosage , cis-trans-Isomerases/deficiency
7.
Equine Vet J ; 44(2): 238-43, 2012 Mar.
Article in English | MEDLINE | ID: mdl-21696432

ABSTRACT

Presently, intraocular lenses (IOLs) are not routinely implanted after equine cataract surgery. Subsequently, horses are visual but markedly farsighted (hyperopic). This report describes the surgical results and visual status after phacoemulsification and implantation of IOLs in mature horses with spontaneous cataracts. Six eyes of 5 mature horses underwent phacoemulsification and implantation of a +14 diopter (D) foldable IOL. Recheck ocular examinations were performed at 1, 4 and 24 weeks post operatively. Refractive error was recorded at 4 weeks post operatively. Visual status, refractive error and anterior chamber depth were recorded 24 weeks post operatively: 5 of 6 operated eyes remained visual and the average refractive error was +0.4 ± 1.1D. There was a significant difference between the 24 week post operative refractive error and the population mean of +10D (P<0.0001) for aphakic horses. The average post operative anterior chamber depth was 7.89 ± 1.55 mm. One globe was enucleated 2 months post operatively. Phacoemulsification with IOL implantation resulted in a significant reduction in post operative refractive error and restored vision to within 0.4D of emmetropia in 5 of 6 operated eyes. Implantation of a +14D IOL ameliorated the hyperopia documented in aphakic horses and improved the post operative visual acuity.


Subject(s)
Cataract/veterinary , Lens Implantation, Intraocular/veterinary , Lenses, Intraocular/veterinary , Phacoemulsification/veterinary , Animals , Female , Horses , Lens Implantation, Intraocular/methods , Male , Phacoemulsification/methods
8.
Gene Ther ; 18(1): 53-61, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20703309

ABSTRACT

The purpose of this study was to evaluate whether immune responses interfered with gene therapy rescue using subretinally delivered recombinant adeno-associated viral vector serotype 2 carrying the RPE65 cDNA gene driven by the human RPE65 promoter (rAAV2.hRPE65p.hRPE65) in the second eye of RPE65-/- dogs that had previously been treated in a similar manner in the other eye. Bilateral subretinal injection was performed in nine dogs with the second eye treated 85-180 days after the first. Electroretinography (ERG) and vision testing showed rescue in 16 of 18 treated eyes, with no significant difference between first and second treated eyes. A serum neutralizing antibody (NAb) response to rAAV2 was detected in all treated animals, but this did not prevent or reduce the effectiveness of rescue in the second treated eye. We conclude that successful rescue using subretinal rAAV2.hRPE65p.hRPE65 gene therapy in the second eye is not precluded by prior gene therapy in the contralateral eye of the RPE65-/- dog. This finding has important implications for the treatment of human LCA type II patients.


Subject(s)
Carrier Proteins/genetics , Eye Proteins/genetics , Genetic Therapy/methods , Retina/physiopathology , Animals , Carrier Proteins/metabolism , Dependovirus/genetics , Dependovirus/metabolism , Dogs , Electroretinography , Eye Proteins/metabolism , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Genetic Vectors/metabolism , Humans , Immunoglobulin G/metabolism , Immunoglobulin M/metabolism , Leber Congenital Amaurosis/physiopathology , Leber Congenital Amaurosis/therapy , cis-trans-Isomerases
9.
Mol Vis ; 15: 1835-42, 2009 Sep 11.
Article in English | MEDLINE | ID: mdl-19756181

ABSTRACT

PURPOSE: To compare self-complementary (sc) and single-stranded (ss) adeno-associated viral 2/5 (AAV2/5) vectors for retinal cell transduction in the dog when delivered by subretinal injection. METHODS: ScAAV2/5 and ssAAV2/5 vectors encoding enhanced green fluorescent protein (GFP) under control of the chicken beta actin promoter were prepared to the same titer. Equal amounts of viral particles were delivered into the subretinal spaces of both eyes of two dogs. In each dog, one eye received the scAAV2/5 and the other the ssAAV2/5. In vivo expression of GFP was monitored ophthalmoscopically. The dogs were sacrificed, and their retinas were examined by fluorescent microscopy and immunohistochemistry to determine GFP expression patterns and to assay for glial reactivity. RESULTS: GFP expression in the scAAV2/5 injected eyes was detectable at a much earlier time point than in the ssAAV2/5 injected eyes. Expression of GFP was also at higher levels in the scAAV2/5-injected eyes. Expression levels remained stable for the seven month duration of the study. The types of cells transduced by both vectors were similar; there was strong reporter gene expression in the RPE and photoreceptors, although not all cones in the transduced area expressed GFP. Some horizontal and Müller cells were also transduced. CONCLUSIONS: When delivered by subretinal injection in the dog, scAAV2/5 induces faster and stronger transgene expression than ssAAV2/5. The spectrum of retinal neurons transduced is similar between the two vectors. These results confirm in a large animal model those previously reported in the mouse. ScAAV2/5 shows promise for use in the treatment of conditions where a rapid transgene expression is desirable. Furthermore, it may be possible to use a lower number of viral particles to achieve the same effect compared with ssAAV2/5 vectors.


Subject(s)
DNA, Complementary/genetics , Dependovirus/genetics , Genetic Vectors/genetics , Transduction, Genetic , Animals , Dogs , Female , Glial Fibrillary Acidic Protein , Green Fluorescent Proteins/metabolism , Immunohistochemistry , Male , Models, Animal , Neuroglia/cytology , Retina/cytology
10.
J Virol ; 75(20): 9885-95, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11559821

ABSTRACT

The highly conserved coadapters CREB binding protein (CBP) and p300 form complexes with CREB as well as other DNA binding transcription factors to modulate chromatin remodeling and thus transcription. Human T-lymphotropic virus type 1 (HTLV-1) transcription is controlled, in part, by the CREB/ATF family of transcription factors which bind promoter sequences and function as complexes with the viral oncogenic protein Tax. We have reported that the nuclear localizing protein p30(II) of HTLV-1 functions as a transcription factor, differentially modulates CREB-responsive promoters, and is critical for maintenance of proviral loads in rabbits. In this study, we tested whether p30(II) directly interacts with CBP/p300 to modulate gene transcription. Gal4(BD)-p30(II)-mediated transactivation was enhanced following exogenous expression of p300 and was competitively repressed by the p300 binding protein, adenovirus E1A, and E1ACR2 (mutated for retinoblastoma binding but retaining p300 binding). In contrast, E1ACR1 (mutated for p300 binding) failed to alter Gal4(BD)-p30(II)-mediated transactivation. In addition, Gal4(BD)-p30(II)-mediated transactivation was competitively inhibited by the cotransfection of CMV-p30(II)-HA and CMV-Tax but could be rescued by exogenous p300. Importantly, we demonstrate that p30(II) colocalizes with p300 in cell nuclei and directly binds to CBP/p300 in cells. Deletion mutants of CBP/p300 were used to localize the site critical for binding p30(II) to a highly conserved KIX region. DNA binding assays confirmed the interference of p30(II) with the assembly of CREB-Tax-p300/CBP multiprotein complexes on 21-bp repeat oligonucleotides in vitro. Collectively, our results demonstrate that CBP/p300 is a cellular protein target for HTLV-1 p30(II) and mediates its transcriptional effects in vivo.


Subject(s)
Human T-lymphotropic virus 1/physiology , Nuclear Proteins/metabolism , Retroviridae Proteins/metabolism , Trans-Activators/metabolism , Transcription, Genetic , Adenovirus E1A Proteins/genetics , Adenovirus E1A Proteins/metabolism , Cell Line , Cyclic AMP Response Element-Binding Protein/metabolism , Human T-lymphotropic virus 1/genetics , Human T-lymphotropic virus 1/metabolism , Humans , Mutation , Nuclear Proteins/genetics , Protein Binding , Trans-Activators/genetics
11.
J Virol ; 74(23): 11270-7, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11070026

ABSTRACT

Human T-lymphotropic virus type 1 (HTLV-1), a complex retrovirus, causes adult T-cell lymphoma/leukemia and is linked to a variety of immune-mediated disorders. The roles of proteins encoded in the pX open reading frame (ORF) II gene region in HTLV-1 replication or in mediating virus-associated diseases remain to be defined. A nucleus-localizing 30-kDa protein, p30(II), encoded within pX ORF II has limited homology with the POU family of transcription factors. Recently, we reported that selected mutations in pX ORF II diminish the ability of HTLV-1 to maintain high viral loads in infected rabbits. Herein we have tested the transcriptional ability of p30(II) in mammalian cells by using yeast Gal4 fusion protein vectors and transfection of luciferase reporter genes driven by CREB-responsive promoters. p30(II) as a Gal4 DNA-binding domain (DBD) fusion protein transactivates Gal4-driven luciferase reporter gene activity up to 25-fold in 293 and HeLa-tat cells. We confirmed nuclear localization of p30(II) and demonstrate dose-dependent binding of p30(II)-Gal4(DBD) to Gal4 DNA-binding sites. The transcriptional activity of p30(II)-Gal4(DBD) was independent of TATA box flanking sequences, as shown by using two different Gal4 reporter systems. Studies of selected p30(II) mutants indicated that domains that mediate transcription are restricted to a central core region of the protein between amino acids 62 and 220. Transfection of a p30(II)-expressing plasmid repressed cellular CRE-driven reporter gene activity, with or without Tax expression. In contrast, p30(II) at lower concentrations enhanced HTLV-1 long terminal repeat-driven reporter gene activity independent of Tax expression. These data are the first to demonstrate a transcriptional function for p30(II) and suggest a mechanism by which this nuclear protein may influence HTLV-1 replication or cellular gene expression in vivo.


Subject(s)
Cyclic AMP Response Element-Binding Protein/pharmacology , Human T-lymphotropic virus 1/genetics , Promoter Regions, Genetic , Transcription Factors/physiology , Viral Proteins/physiology , Animals , Cell Nucleus/chemistry , Gene Expression Regulation , HeLa Cells , Humans , Open Reading Frames , Rabbits , Response Elements , Terminal Repeat Sequences , Transcriptional Activation , Viral Proteins/analysis , Virus Replication
12.
AIDS Res Hum Retroviruses ; 16(16): 1757-64, 2000 Nov 01.
Article in English | MEDLINE | ID: mdl-11080823

ABSTRACT

Human T lymphotropic virus type 1 (HTLV-1) is a complex retrovirus containing regulatory and accessory genes encoded in four open reading frames (ORF I-IV) of the pX region. It is not clear what role pX ORFs I and II-encoded proteins have in the pathogenesis of the lymphoproliferative diseases associated with HTLV-1 infection. The conserved ORF I encodes for a hydrophobic 12-kDa protein, p12, (I) that contains four SH3 binding motifs (PXXP) that localizes to cellular endomembranes when overexpressed in cultured cells. Differential splicing of pX ORF II results in the production of two nuclear proteins, p13(II) and p30(II). p13(II) also localizes to mitochondria. p30(II) shares homology with the POU family of transcription factors. We have identified functional roles of pX ORF I and ORF II in establishment and maintenance of infection in a rabbit model. To functionally study p12(I) we have tested a proviral clone with selective ablation of ORF I (ACH.p12(I)) for its ability to infect quiescent peripheral blood mononuclear cells (PBMC). Our data indicate that T cells infected with the wild-type clone of HTLV-1 (ACH) are more efficient than ACH.p12(I) in infecting quiescent PBMC. These findings parallel our animal model data and suggest a role for p12(I) in the activation of quiescent lymphocytes, a prerequisite for effective viral replication in vivo. To test the ability of p30(II) to function as a transcription factor we have constructed p30(II) as a Gal4-fusion protein. When transfected with Gal4-driven luciferase reporter genes, the p30(II)-Gal4-fusion protein induces transcriptional activity up to 50-fold in both 293 and HeLa-Tat cells. These systems will be useful to identify molecular mechanisms that explain the functional role of pX ORF I and ORF II-encoded proteins in HTLV-1 replication.


Subject(s)
Genes, pX/genetics , Genes, pX/physiology , Human T-lymphotropic virus 1/immunology , Human T-lymphotropic virus 1/pathogenicity , Open Reading Frames , Transcription Factors , Animals , Cell Transformation, Viral , HTLV-I Infections/virology , Human T-lymphotropic virus 1/genetics , Humans , Leukocytes, Mononuclear/virology , Lymphocyte Activation , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism , Rabbits , Retroviridae Proteins/genetics , Retroviridae Proteins/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/virology , Transcriptional Activation , Viral Regulatory and Accessory Proteins , Virus Replication
13.
J Virol ; 74(3): 1094-100, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10627519

ABSTRACT

Human T-lymphotropic virus type 1 (HTLV-1) causes adult T-cell leukemia/lymphoma and is associated with a variety of immune-mediated disorders. The role of four open reading frames (ORFs), located between env and the 3' long terminal repeat of HTLV-1, in mediating disease is not entirely clear. By differential splicing, ORF II encodes two proteins, p13(II) and p30(II), both of which have not been functionally defined. p13(II) localizes to mitochondria and may alter the configuration of the tubular network of this cellular organelle. p30(II) localizes to the nucleolus and shares homology with the transcription factors Oct-1 and -2, Pit-1, and POU-M1. Both p13(II) and p30(II) are dispensable for infection and immortalization of primary human and rabbit lymphocytes in vitro. To test the role of ORF II gene products in vivo, we inoculated rabbits with lethally irradiated cell lines expressing the wild-type molecular clone of HTLV-1 (ACH.1) or a clone containing selected mutations in ORF II (ACH.30/13.1). ACH.1-inoculated animals maintained higher HTLV-1-specific antibody titers than animals inoculated with ACH.30/13.1. Viral p19 antigen was transiently detected in ex vivo cultures of peripheral blood mononuclear cells (PBMC) from only two ACH.30/13.1-inoculated rabbits, while PBMC cultures from all ACH.1-inoculated rabbits routinely produced p19 antigen. In only three of six animals exposed to the ACH. p30(II)/p13(II) clone could provirus be consistently PCR amplified from extracted PBMC DNA and quantitative competitive PCR showed the proviral loads in PBMC from ACH.p30(II)/p13(II)-infected rabbits to be dramatically lower than the proviral loads in rabbits exposed to ACH. Our data indicate selected mutations in pX ORF II diminish the ability of HTLV-1 to maintain high viral loads in vivo and suggest an important function for p13(II) and p30(II) in viral pathogenesis.


Subject(s)
HTLV-I Infections/virology , Human T-lymphotropic virus 1/genetics , Human T-lymphotropic virus 1/physiology , Open Reading Frames/genetics , Viral Load , Animals , Blotting, Western , Cell Line , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Gene Products, gag/blood , HTLV-I Antibodies/blood , Human T-lymphotropic virus 1/isolation & purification , Humans , Leukocytes, Mononuclear/virology , Mutation , Polymerase Chain Reaction , Proviruses , Rabbits , Retroviridae Proteins, Oncogenic/blood , gag Gene Products, Human Immunodeficiency Virus
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