Correction of acid beta-galactosidase deficiency in GM1 gangliosidosis human fibroblasts by retrovirus vector-mediated gene transfer: higher efficiency of release and cross-correction by the murine enzyme

Mutations in the lysosomal acid beta-galactosidase (EC 3.2.1.23) underlie two different disorders: GM1 gangliosidosis, which involves the nervous system and visceral organs to varying extents, and Morquio's syndrome type B (Morquio B disease), which is a skeletal-connective tissue disease without any CNS symptoms. This article shows that transduction of human GM1 gangliosidosis fibroblasts with retrovirus vectors encoding the human acid beta-galactosidase cDNA leads to complete correction of the enzymatic deficiency. The newly synthesized enzyme is correctly processed and targeted to the lysosomes in transduced cells. Cross-correction experiments using retrovirus-modified cells as enzyme donors showed, however, that the human enzyme is transferred at low efficiencies. Experiments using a different retrovirus vector carrying the human cDNA confirmed this observation. Transduction of human GM1 fibroblasts and mouse NIH 3T3 cells with a retrovirus vector encoding the mouse beta-galactosidase cDNA resulted in high levels of enzymatic activity. Furthermore, the mouse enzyme was found to be transferred to human cells at high efficiency. Enzyme activity measurements in medium conditioned by genetically modified cells suggest that the human beta-galactosidase enzyme is less efficiently released to the extracellular space than its mouse counterpart. This study suggests that lysosomal enzymes, contrary to the generalized perception in the field of gene therapy, may differ significantly in their properties and provides insights for design of future gene therapy interventions in acid beta-galactosidase deficiency.     

Characterization of alpha-L-fucosidase from two different families with fucosidosis.

1. Two different families with a different type of fucosidase deficiency are described. 2. In the first family the activity of alpha-L-fucosidase in leucocytes of two patients with fucosidosis type I was about 4 to 8% of the normal value. The activity of alpha-L-fucosidase in the leucocytes of the father and the mother are in the heterozygote range, while a sister of the propositus showed normal values. 3. The activity of alpha-L-fucosidase of the propositus from urine, serum and liver were also severely decreased. The activity of alpha-L-fucosidase in the urine of the parents and the healthy sister of thr propositus were about 5% of the mean normal value. However in the serum these values were above 50%. 4. The KM value for alpha-L-fucosidase from leucocytes of the patient was increased about 10 times and in serum this value was even higher. The KM values from the enzyme of the parents were in the normal range. 5. The abnormal enzyme from the propositus is unique in its thermal behaviour since after heating its activity increased. 6. In the second fanily the activity of alpha-L-fucosidase in the leucocytes of the patient is about 30% of the mean normal value, while the arylsulphatase A activity is also decreased (25% of the mean normal value). 7. The activity of alpha-L-fucosidase from the leucocytes of the father and the healthy brother are about 50% of the mean normal level, while the enzyme of the mother showed a normal activity. 8. The alpha-L-fucosidase activity in the urine and the liver of the propositus is also decreased. The serum enzyme activity however was in the normal range. 9. The KM value of alpha-L-fucosidase and heat stability of the enzyme of the patient were normal. In the leectrophoretic pattern of the whole family one bond was missing.     

Defective expression of alpha-L-fucosidase by lymphoid cells of a fucosidosis patient

Fucosidosis is an inherited lysosomal storage disease due to a deficiency of alpha-L-fucosidase activity. Exponentially growing lymphoid cell cultures from a fucosidosis patient (JH) had 16-fold lower extracellular alpha-L-fucosidase protein and 72-fold lower intracellular alpha-L-fucosidase protein with negligible catalytic activity as compared with the mean of 19 control cultures. The percentage of total alpha-L-fucosidase protein released extracellularly by JH cells was 71% as compared with 35% +/- 9% for control cells. During a 1.5 h pulse with 35S-methionine, alpha-L-fucosidase was synthesized by JH cells as an intracellular doublet with Mr of 58,000 and 56,000 and by control cells as an intracellular form with Mr = 58,000. During a subsequent 21 h chase with unlabeled methionine, JH alpha-L-fucosidase was entirely secreted. In contrast, only 25%-30% of control enzyme was secreted with the remainder retained intracellularly. Thus, JH lymphoid cells synthesized a reduced amount of alpha-L-fucosidase that was catalytically inefficient and was hypersecreted. Treatment of JH alpha-L-fucosidase with N-glycanase produced polypeptide chains with Mr of 52,000 and 54,000. Previously, treatment of control alpha-L-fucosidase with N-glycancase produced a single polypeptide chain with Mr of 52,000 (Biochem Genet 1988; 26: 401-20). The doublet polypeptide chains of alpha-L-fucosidase in JH cultures may represent expression of two distinct allelic forms of mutant alpha-L-fucosidase.     

CAR-dependent and CAR-independent pathways of adenovirus vector-mediated gene transfer and expression in human fibroblasts

Primary fibroblasts are not efficiently transduced by subgroup C adenovirus (Ad) vectors because they express low levels of the high-affinity Coxsackie virus and adenovirus receptor (CAR). In the present study, we have used primary human dermal fibroblasts as a model to explore strategies by which Ad vectors can be designed to enter cells deficient in CAR. Using an Ad vector expressing the human CAR cDNA (AdCAR) at high multiplicity of infection, primary fibroblasts were converted from being CAR deficient to CAR sufficient. Efficiency of subsequent gene transfer by standard Ad5-based vectors and Ad5-based vectors with alterations in penton and fiber was evaluated. Marked enhancement of binding and transgene expression by standard Ad5 vectors was achieved in CAR-sufficient fibroblasts. Expression by AdDeltaRGDbetagal, an Ad5-based vector lacking the arginine-glycine-aspartate (RGD) alphaV integrin recognition site from its penton base, was achieved in CAR-sufficient, but not CAR-deficient, cells. Fiber-altered Ad5-based vectors, including (a) AdF(pK7)betagal (bearing seven lysines on the end of fiber) (b) AdF(RGD)betagal (bearing a high-affinity RGD sequence on the end of fiber), and (c) AdF9sK betagal (bearing a short fiber and Ad9 knob), demonstrated enhanced gene transfer in CAR-deficient fibroblasts, with no further enhancement in CAR-sufficient fibroblasts. Together, these observations demonstrate that CAR deficiency on Ad targets can be circumvented either by supplying CAR or by modifying the Ad fiber to bind to other cell-surface receptors.     

Correction of mucopolysaccharidosis type I fibroblasts by retroviral-mediated transfer of the human alpha-L-iduronidase gene.

Three retroviral constructs containing a full-length human alpha-L-iduronidase (IDUA) cDNA were made. The first, pLIdSN, is designed so that expression of the IDUA cDNA is from the 5' viral long terminal repeat (LTR). The second, pLNCId, is designed to express the IDUA cDNA from the cytomegalovirus (CMV) immediate early promoter, while in the third, pLNTId, the CMV promoter is replaced by a promoter fragment of the mouse CD45 (T200) gene. All vectors transduce resistance to G418 (neomycin). High-titer virus-producing cell lines for these constructs were made by infection of the amphotropic packaging cell line PA317 after transient expression in, and virus rescue from, the ecotropic packaging cell line psi CRE. The high-titer virus-producing cell lines were assayed for absence of helper virus, synthesis of human IDUA, and for integrity of proviral structure. Suitable lines were used as a source of virus to infect two different mucopolysaccharidosis type I (MPS I) skin fibroblast cultures. All three of the recombinant viruses corrected the enzymatic defect in MPS I fibroblasts. Surprisingly, increasing over-expression of IDUA resulted in reduced phenotypic correction of these cells as assayed by intracellular accumulation of 35S-labeled glycosaminoglycan. This was shown to be due to the induction of a phenotype analogous to mild I-cell disease in cells expressing large amounts of IDUA.     

Purification and serological studies of human alpha-L-fucosidase in the normal and fucosidosis states.

An antiserum has been raised to purified alpha-L-fucosidase. Levels of cross-reaction with serum of two unrelated fucosidosis patients and normal individuals with low activity are consistent with the presence of very low amounts of normal enzyme. Similarly no cross-reacting material could be found in cultured fibroblasts from fucosidosis patients. It is deduced that in these cases there is no production of mutant enzyme in quantities comparable to normal levels. Some observations on the interrelations of fucosidases I and II are reported.     

Efficient in vivo xenogeneic retroviral vector-mediated gene transduction into human hepatocytes

We developed a method for efficient retroviral vector-mediated gene transfer into human hepatocytes, using a human hepatocyte-bearing mouse model. Normal human hepatocytes were transplanted into the livers of immunodeficient and liver-damaged mice. Donor hepatocytes multiplied and replaced the host hepatocytes, which yielded human hepatocyte-bearing mice (human hepatocyte-chimeric mice). As control cells, rat hepatocytes were similarly transplanted. The replacement level reached 86% at 8 weeks and 100% at 5 weeks posttransplantation of human and rat hepatocytes, respectively. Human and rat hepatocytes in the host liver showed a high bromodeoxyuridine-labeling index during the first 2 weeks posttransplantation. Human- and rat-chimeric mice were injected 7 and 10 days posttransplantation, respectively, with retroviral vectors carrying the beta-galactosidase gene and were thereafter injected daily for 20 and 10 days, respectively. The level of beta-galactosidase-positive hepatocytes in the human- and rat-chimeric mice reached 7.1 +/- 1.8% at 8 weeks and 5.3 +/- 0.9% at 5 weeks after transplantation, respectively. The human hepatocyte-chimeric mouse will be useful for testing the ability of vectors to transduce human cells.     

Improvements in mucopolysaccharidosis I mice after adult retroviral vector-mediated gene therapy with immunomodulation.

Mucopolysaccharidosis I (MPS I) is caused by deficient alpha-L-iduronidase (IDUA) activity and results in the accumulation of glycosaminoglycans and multisystemic disease. Gene therapy could program cells to secrete mannose 6-phosphate-modified IDUA, and enzyme in blood could be taken up by other cells. Neonatal retroviral vector (RV)-mediated gene therapy has been shown to reduce the manifestations of murine MPS I; however, intravenous injection of RV into adults was ineffective owing to a cytotoxic T lymphocyte (CTL) response against transduced cells. In this study, prolonged inhibition of CD28 signaling with CTLA4-Ig, or transient administration of CTLA4-Ig with an anti-CD40 ligand antibody or with an anti-CD4 antibody, resulted in stable expression in most mice that received RV as adults. Mice with stable expression had 81 +/- 41U/ml IDUA activity in serum. This resulted in reductions in bone disease, improvements in hearing and vision, and reductions in biochemical and pathological evidence of lysosomal storage in most organs. Improvements in brain were likely due to diffusion of enzyme from blood. However, aortic disease was refractory to treatment. This demonstrates that most manifestations of MPS I can be prevented using adult gene therapy if an immune response is blocked.     

Retrovirus vector-mediated correction and cross-correction of lysosomal alpha-mannosidase deficiency in human and feline fibroblasts.

Lysosomal alpha-mannosidase (EC 3.2.1.24) is an exoglycosidase in the glycoprotein degradation pathway. A deficiency of this enzyme causes the lysosomal storage disease alpha-mannosidosis. Retrovirus vector transfer of a new human alpha-mannosidase cDNA resulted in high-level expression of alpha-mannosidase enzymatic activity in deficient human and feline fibroblasts. The expressed alpha-mannosidase had the same biochemical properties (thermal stability, pH profile, inhibitor/activator sensitivity) as the native enzyme expressed in normal cells. The transferred enzyme colocalized with a control lysosomal hydrolase in cell fractionation experiments. The vector-encoded enzyme also was released at high levels from the corrected cells, and was taken up by untreated mutant cells via the mannose 6-phosphate receptor-mediated endocytic pathway (cross-correction). It is envisioned that genetic correction of a subset of cells (e.g., hematopoietic stem cells) in patients will provide a source of corrective enzyme for other affected tissues in this multisystem disease. Development of a vector expressing high levels of alpha-mannosidase that cross-corrects mutant cells will enable somatic gene transfer experiments in the cat model of human alpha-mannosidosis.     

High efficiency myogenic conversion of human fibroblasts by adenoviral vector-mediated MyoD gene transfer. An alternative strategy for ex vivo gene therapy of primary myopathies.

Ex vivo gene therapy of primary myopathies, based on autologous transplantation of genetically modified myogenic cells, is seriously limited by the number of primary myogenic cells that can be isolated, expanded, transduced, and reimplanted into the patient's muscles. We explored the possibility of using the MyoD gene to induce myogenic conversion of nonmuscle, primary cells in a quantitatively relevant fashion. Primary human and murine fibroblasts from skin, muscle, or bone marrow were infected by an E1-deleted adenoviral vector carrying a retroviral long terminal repeat-promoted MyoD cDNA. Expression of MyoD caused irreversible withdrawal from the cell cycle and myogenic differentiation in the majority (from 60 to 90%) of cultured fibroblasts, as defined by activation of muscle-specific genes, fusion into contractile myotubes, and appearance of ultrastructurally normal sarcomagenesis in culture. 24 h after adenoviral exposure, MyoD-converted cultures were injected into regenerating muscle of immunodeficient (severe combined immunodeficiency/beige) mice, where they gave rise to beta-galactosidase positive, centrally nucleated fibers expressing human myosin heavy chains. Fibers originating from converted fibroblasts were indistinguishable from those obtained by injection of control cultures of lacZ-transduced satellite cells. MyoD-converted murine fibroblasts participated to muscle regeneration also in immunocompetent, syngeneic mice. Although antibodies from these mice bound to adenoviral infected cells in vitro, no inflammatory infiltrate was present in the graft site throughout the 3-wk study period. These data support the feasibility of an alternative approach to gene therapy of primary myopathies, based on implantation of large numbers of genetically modified primary fibroblasts massively converted to myogenesis by adenoviral delivery of MyoD ex vivo.     

Therapeutic levels of human protein C in rats after retroviral vector-mediated hepatic gene therapy.

Protein C deficiency results in a thrombotic disorder that might be treated by expressing a normal human protein C (hPC) gene in patients. An amphotropic retroviral vector with a liver-specific promoter and the hPC cDNA was delivered to rat hepatocytes in vivo during liver regeneration. Expression of hPC varied from 55 to 203 ng/ml (1.3-5.0% of normal) for 2 wk after transduction. Expression increased to an average of 900 ng/ml (22% of normal) in some rats and was maintained at stable levels for 1 yr. All of these rats developed anti-hPC antibodies and exhibited a prolonged hPC half-life in vivo. The hPC was functional as determined by a chromogenic substrate assay after immunoprecipitation. We conclude that most rats achieved hPC levels that would prevent purpura fulminans, and that hepatic gene therapy might become a viable treatment for patients with severe homozygous hPC deficiency. Anti-hPC antibodies increased the hPC half-life and plasma levels in some rats, but did not interfere with its functional activity. Thus, the development of antibodies against a plasma protein does not necessarily abrogate its biological effect in gene therapy experiments.     

Viral vector-mediated gene transfer for CNS disease

Importance of the field: Gene therapy is a promising strategy for the treatment of many neurological disorders that currently lack effective treatment. Recent improvements in vectorology and vector engineering have improved overall safety and delivery of viral vectors.

Areas covered in this review: This review discusses the current state of viral vector development and clinical use, as well as routes of delivery, and clinical trials for neurological disorders.

What the reader will gain: Viral vectors may be delivered directly or remotely to the CNS, largely depending on the nature of the disease and the tropism of the vector. Nonetheless, delivery remains one of the major limitations of successful gene transfer to the CNS.

Take home message: Although the majority of clinical trials have centered on gene replacement and neuroprotection approaches, the field is advancing in the direction of neuromodulation, gene silencing and other newer strategies.     

Efficient gene transfer by hybrid retroviral vectors to murine spermatogenic cells.

Using murine spermatogenic cell lines GC-1 spg and GC-2 spd(ts) as target cells, an attempt was made to design a retroviral vector that would transduce genes efficiently. Promoter activities of various retroviral long terminal repeats (LTRs) were examined by using chloramphenicol acetyltransferase (CAT) as a reporter. The U3 region of spleen focus-forming virus (SFFVp) showed higher enhancer activity than that of Moloney murine leukemia virus (Mo-MuLV) in both cell lines. The U3 region of myeloproliferative sarcoma virus (MPSV) showed higher activity only in GC-1 spg cells. Expression was suppressed by the repressor element of the primer-binding site (PBS) of the Moloney-related virus. The efficiency of transduction of the multidrug-resistance gene (mdr-1) by an Mo-MuLV-based vector was compared with hybrid vectors consisting of the murine embryonic stem cell virus (MESV) PBS and the LTR of either SFFVp or MPSV. Rhodamine efflux assays and colchicine-resistant colony-forming assays demonstrated higher gene expression by the hybrid vectors. Amphotropic and ecotropic receptors were found to be expressed and functional in both cell lines. Thus, these hybrid vectors represent a powerful tool by which to transfer genes into spermatogenic cells.     

Helper-dependent adenoviral vector-mediated gene transfer in aged rat brain

Transfer of the neurotrophin gene into brain can attenuate age-related deficits such as neuronal atrophy and memory loss, but a suitable vector for this procedure has been lacking. The toxicity and immunogenicity of first-generation adenoviral vectors with E1 deletion (fgAdv) prohibit the application of gene transfer in the majority of central nervous system disorders. Here, we report less toxic and persistent gene expression mediated by helper-dependent adenovirus (hdAdv) in aged rat brain. After intrahippocampal or intraventricular inoculation of the vector, transgene expression was monitored by X-Gal staining and compared with fgAdv-mediated expression. Host inflammatory and immune responses against these vectors were evaluated by immunohistochemical detection of microglia, astrocytes, and infiltrating macrophages, as well as by enzyme-linked immunosorbent assay of cytokines TNF-alpha and IL-1beta. Transgene expression mediated by hdAdv persisted for more than 183 days regardless of inoculation site, as compared with 33 and 66 days for fgAdv-mediated expression after intraventricular and intrahippocampal inoculation, respectively. Inoculation with hdAdv was also associated with reduced numbers of activated microglial cells, astrocytes, and infiltrating macrophages in brain tissue. Secretion of the proinflammatory cytokines TNF-alpha and IL-1beta was minimal after hdAdv but not after fgAdv inoculation. These findings indicate that hdAdv would provide a safe and effective means to transfer therapeutic genes into aged brain.     

Adenovirus vector-mediated gene transfer to regional lymph nodes

Regional lymph nodes (RLNs) possess important immune functions and represent a major pathway of metastasis for solid tumors. Given these facts, the ability to transfer exogenous genes to the RLNs with the goal of manipulating the local immunological milieu would be desirable. On the basis of the hypothesis that a significant proportion of adenovirus (Ad) gene transfer vectors traffic through the lymphatics, E1-E3- Ad vectors were injected into the hind footpad of C3H/He mice and the RLNs assessed for vector trafficking and transgene expression. A low dose (10(9) particles) of an Ad vector encoding the firefly luciferase gene (Ad-CMV.Luc) resulted in luciferase expression only in the injection site and RLNs, with no detectable systemic (liver, spleen, lung) expression. At a higher dose (10(11) particles), some expression could be detected systemically in addition to the RLNs, but at levels in liver 14-fold less than in the RLNs. Transgene expression in the RLNs was transient, peaking at 1 day, decreasing markedly by 7 days. At high doses (10(11) particles), interruption of draining lymphatics decreased the amount of systemic dissemination 22-fold, suggesting that a large proportion of the vector trafficks through the lymphatics before reaching the systemic circulation. Administration of a vector encoding the jellyfish green fluorescent protein gene (AdCMV.GFP, 10(11) particles) showed that transgene expression in the RLNs was primarily in the cortical area. After footpad injection of a fluorescent-labeled Ad vector (Cy3-AdCMV.Null), fluorescent virions were visualized in the draining lymph. Regional lymph collected from animals injected in the footpad with AdCMV.Luc (10(11) particles) contained functional vector. Augmentation of local immune function in the RLNs was achieved by footpad administration of an Ad vector encoding murine IL-12, resulting in high mIL-12 and IFN-gamma levels in the regional, but not distant, nodes. These data demonstrate that expression of exogenous genes in RLNs is easily accomplished with Ad vectors, Ad vector dissemination occurs primarily via the lymphatics after footpad administration in mice, and basic immune functions in the RLNs can be manipulated by Ad-mediated gene transfer in vivo.     

反转录病毒载体介导转化生长因子β_1基因体外转染兔膝关节软骨细胞的表达

背景:将功能基因片段整合到基因载体内,再将基因载体转染入靶细胞中或转染入关节腔内,通过转基因的靶细胞持续大量分泌功能基因产物,在一个较长的时期内保持局部治疗浓度,可修复关节软骨损伤.目的:以重组反转录病毒载体介导转化生长因子β_1体外转染兔膝关节软骨细胞,观察其表达情况及其对软骨细胞生物学性状的影响.方法:采用胰酶消化法分离培养兔软骨细胞.载体经PLNCX_2 Hind Ⅲ/Not Ⅰ双酶切、去磷酸化后,与载体pDsRed_2双酶切得到大的部分多克隆位点和RFP基因连接,构建PLNCX_2-RFP.转化生长因子β_1基因从PGEMT-TGF中扩增,经双酶切后与PLNCX_2-RFP连接,构建PLNCX_2-TGFβ_1-RFP.包装反转录病毒载体,并检测病毒上清滴度.将培养的兔膝关节软骨细胞分组转染:对照组(不做任何转染)、转染PLNCX_2组、转染PLNCX_2-TGFβ_1-RFP组,持续筛选2周,观察细胞变化.收集稳定转染的细胞上清液,以NO检测试剂盒检测基因转染对软骨细胞的影响,以ELISA法检测细胞培养上清液中人转化生长因子β_1表达.结果与结论:重组基因PLNCX_2-TGFβ_1-RFP经酶切鉴定测序TGFβ_1、RFP、序列均正确,表明构建成功预期的真核表达载体PLNCX_2-TGFβ_1-RFP.转染到包装细胞并筛选培养后,病毒滴度为1×10~6CFU.稳定转染软骨细胞后可观察到红色荧光,证明转染成功.持续筛选2周,散在贴壁细胞形成阳性克隆,逐渐弥漫融合,并有细胞簇出现,双核多见,细胞增生活跃.转染PLNCX_2-TGFβ_1-RFP组NO浓度高于对照组、转染PLNCX_2组(P<0.05),对照组、转染PLNCX_2组间差异无显著性意义.对照组与转染PLNCX_2组均无转化生长因子β_1表达,转染PLNCX_2-TGFβ_1-RFP组转化生长因子β_1质量浓度为(28.08±3.73)ng/L.提示反转录病毒载体PLNCX_2介导的人转化生长因子β_1能有效转染到兔膝关节软骨细胞并获得稳定表达,同时转染后的软骨细胞增生活跃.     

Persistent gene expression after retroviral gene transfer into liver cells in vivo.

The development of liver-directed gene therapy protocols depends upon the ability to transfer genes into a large number of liver cells such that the genes are expressed persistently. We used a retroviral vector to transfer the gene for neomycin phosphotransferase (neo) into mouse liver cells in vivo. Direct injection of the retrovirus preparation into mitotically active (regenerating) liver parenchyma resulted in efficient gene transfer, with neo sequences detectable in the livers of every animal tested 10 weeks to 6 months later. The neo gene was expressed for at least 3 months. This methodology may eventually be applicable to the treatment of human disease.     

Comparison of the effects of growth factors on retroviral vector-mediated gene transfer and the proliferative status of human hematopoietic progenitor cells

We have examined the ability of the recombinant hematopoietic growth factors (HGF) interleukin-3 (IL-3), IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) to increase retroviral vector-mediated gene transfer into human hematopoietic progenitor cells (HPC). The efficiency of neo gene transfer by the N2 vector into human HPC was enhanced by preculture with either GM-CSF or IL-3 (but not IL-6) and with each combination of the three factors. The combination of IL-3 plus IL-6 consistently produced significantly higher levels of G418-resistant colonies (50-60%) than any of the other combinations of HGF tested. Following preculture with HGF and transduction by N2, marrow was maintained in long-term bone marrow culture (LTBMC) for 2 months. The levels of G418-resistant HPC remained stable, and no apparent depletion of total HPC content resulted from the prior exposure to highly stimulatory doses of factors. The proliferative status of the HPC, following exposure to the HGF, was measured as the percentage of HPC that were inhibited from forming colonies by exposure to the S-phase-specific drug, hydroxyurea. The ability of the different HGF to increase the rate of gene transfer by N2 correlated significantly with the extent to which they stimulated HPC proliferation. These results suggest that the mechanism by which HGF increase rates of gene transfer into HPC is by stimulating cell proliferation. Techniques that produce high rates of gene transfer into long-lived human HPC will facilitate studies to quantitate expression of exogenous genes in hematopoietic cells and may be applicable to clinical gene therapy.     

Retroviral vector-mediated gene transfer into hematopoietic cells: prospects and issues.

Gene therapy is a developing technology that may allow the treatment of a variety of congenital and acquired genetic disorders as well as infectious diseases through the introduction of exogenous genetic material into relevant cellular populations. Currently, the most effective method for gene transfer into cells of the hematopoietic system is with retroviral vectors. Appropriate cellular targets for gene transfer include totipotent hematopoietic stem cells as well as long-lived lineage committed cells such as T lymphocytes. Although retroviral vector-mediated gene transfer into totipotent stem cells and subsequent long-term expression of transduced genetic material in stem cell progeny has been observed in murine bone marrow transplantation experiments, similar observations have not been made in clinically relevant large-animal models. A number of recent advances in gene delivery systems, purification of stem cells, defining extramedullary sources of stem cells, characterizing the biologic processes that regulate the proliferation and developmental potential of stem cells, and construction of more effective models for assessing stem cells, may result in improvements in gene transfer into large animal and human totipotent stem cells.