Human Gene Therapy

To develop a potential gene therapy strategy for the treatment of hemophilia A, we constructed several retroviral vectors expressing a B-domain-deleted factor VIII (FVIII) cDNA. We confirmed previous reports that when the FVIII cDNA is inserted into a retroviral vector, the vector mRNA is decreased resulting in significantly (100- to 1,000-fold) lower vector titers. In an attempt to overcome this inhibition we pursued two independent strategies. First, site-directed mutagenesis was employed to change the structure of a putative 1.2-kb FVIII RNA inhibitory sequence (INS). Second, the FVIII gene was transcribed from a retroviral vector containing a 5′ intron. Results demonstrated that the intron increased FVIII expression up to 20-fold and viral titer up to 40-fold but conservative mutagenesis of the putative FVIII INS region failed to yield a significant increase in FVIII expression or titer. Using the improved FVIII splicing vector, we transduced a variety of cell types and were able to demonstrate relatively high FVIII expression (10–60 ng of FVIII/10⁶ cells/24 hr). These results underscore the usefulness of these transduced cell types for potential in vivo delivery of FVIII.

We have expanded ovarian tumor-infiltrating lymphocytes (TIL) in low concentrations of recombinant interleukin-2 (rIL-2) to conduct intraperitoneal adoptive immunotherapy trials in patients with ovarian cancer. We have previously demonstrated that certain T cell lines and clones derived from ovarian TIL exhibit in vitro autologous tumor-specific cytotoxicity and/or cytokine production (interferon-γ, tumor necrosis factor-α) preferentially in response to autologous tumor cells. Studies that utilize a marker gene introduced into the DNA of TIL can provide useful information on specific uptake or localization of TIL at tumor sites and on the survival of TIL in vivo. We have conducted a series of preclinical experiments in which we have successfully transfected TIL with G1Na, which encodes the gene for neomycin phosphotransferase (neo^R). Neo^R was detected in at least 10% of CD8⁺ cells (mean = 10.4%) and between 2.5 and 20% of CD4⁺ TIL (mean = 8.5%). Transduction of ovarian TIL with G1Na caused no substantial changes to the T cell phenotypes or in vitro cytotoxicities against ovarian and hematogenous tumor cell targets, or on the rIL-2 requirements of TIL for growth and proliferation. In addition, the intact G1Na provirus in transduced TIL cells was rescuable by replication-competent retrovirus and was transferred into the genome of NIH-3T3 fibroblasts, which were rendered resistant to G418. An enhanced polymerase chain reaction (PCR) procedure utilizing detection by ethidium bromide staining was developed. The enhanced PCR detected 1 in 100,000 neo^R-labeled cells. Furthermore, detection of the G1Na genome in transduced TIL by in situ hybridization with an RNA probe provided evidence for expression of the neo^R gene in transduced TIL. Results obtained from these studies suggest that ovarian TIL-derived T cell lines transduced with the neo^R gene post infection with the G1Na retroviral vector can be utilized to examine the in vivo trafficking pattern of ovarian TIL-derived T cell lines expanded in low concentrations of rIL-2 and their survival.

Despite good initial success in vivo, gene transfer using first-generation replication-defective adenovirus has been reported to lead to transient reporter gene expression and to trigger inflammatory reactions in various organs and animal models. To gain more knowledge on this phenomenon, immune reactions were investigated following in vivo transfection of adult immunocompetent mouse muscle using a ΔE1/E3^a adenoviral vector encoding a β-galactosidase (β-Gal) expression cassette. Cellular and humoral immune reactions, and rejection of β-Gal-positive muscle fibers, occurred within 3 weeks. The muscles showed massive infiltration by macrophages, natural killer cells, and CD8⁺ leukocytes. The mRNA levels of granzyme B and interferon-γ were increased 6 days after vector injection, indicating that the infiltrating lymphocytes were activated. Antibodies were formed against the adenovirus group antigen and the β-Gal gene product 2 weeks after construct injection. The immunosuppressant FK506, however, blocked the cellular infiltration and the humoral response and allowed strong, stable transgene expression over 1 month. These data emphasize the immune problems related to the use of ΔE1/E3^a adenoviruses as vectors for gene therapy, and they underline the potential of FK506 as an immunosuppressant adjunct treatment for adenovirus-mediated gene transfer.

We have investigated the use of column chromatography for the purification of ACN53, a recombinant adenovirus type 5 encoding the human p53 tumor suppressor protein. Anion exchange, size exclusion, hydrophobic interaction, and metal chelating resins were tested; each was found to have distinct advantages and disadvantages. Based on these data, a rapid method was devised for the purification of ACN53. The resultant product was characterized and compared to cesium chloride density-gradient purified virus by SDS-PAGE, Western blot analysis, absorbance spectrum, total particle-to-infectious particle ratio, expression of p53 gene product in Saos-2 cells, growth inhibition of Saos-2 cells, and contamination by ATCC-293 host cell proteins. The results show that column chromatography offers an alternative to ultracentrifugation for the purification of recombinant adenoviruses for use in human gene therapy trials and other research applications.

We have cloned and characterized a 620-bp fragment of DNA that flanks 5′ of the prostate-specific antigen (PSA) gene from a prostate cancer patient. Using DNA transfection, the efficacy of this putative promoter in regulating gene expression was quantitated in several prostate and nonprostate tissue cell lines. Our results demonstrated that the 620-dp DNA fragment actively drives gene expression in LNCaP, a PSA-producing prostate tumor cell line. No promoter activity was detected in the non-PSA-producing prostate tumor lines, DU145 and PC-3, nor in a renal (R11) or breast (MCF-7) cancer cell line. Furthermore, the promoter activity could be regulated in vitro by androgen stimulation. Dihydrotestosterone (DHT) concentrations between 3 and 30 nM induced the highest promoter activity in the transfected LNCaP cells, which parallels the expression profile of the androgen receptor in LNCaP cells. In addition, our PSA promoter exhibited competitive inhibition of the endogenous genomic PSA promoter in transfected LNCaP cells, suggesting that prostate cell-specific DNA-binding proteins are required to activate the PSA promoter. A cytomegalovirus IE1 promoter (CMV promoter) attached to the 5′-flanking region of the PSA promoter increased its potency four-to five-fold while retaining tissue specificity. Our data suggest that a strong tissue-specific negative regulatory element capable of overriding the nonspecific CMV promoter is present in the PSA promoter and confers its tissue specificity. The use of a highly specific promoter-driven gene vector will allow selective expression of therapeutic genes within PSA-producing prostate cancer cells, providing a unique strategy for prostate cancer gene therapy.

Experimental models of vaccination with tumor cells engineered to produce interleukin-4 (IL-4) have shown that the local release of this cytokine is associated with the development of antitumor immunity that may induce regression of established cancer. The aim of this study was to transduce a human melanoma cell line with the gene coding for human IL-4, and to analyze cytokine production, phenotypic characteristics, and antigen expression after transduction. A retroviral vector, constructed by inserting IL-4 cDNA into the LXSN vector, was used to infect the human melanoma cell line Me14932, known to express the MHC class I HLA-A2 and the melanoma-associated antigen Melan-A/MART-1, recognized by HLA-A2-restricted T-cells. The confluence of all G418-resistant cells (Me14932/IL-4) was then analyzed for proviral integration and IL-4 mRNA expression. Substantially stable IL-4 release was detected by ELISA in the supernatant of transduced cells, ranging from 1.6 to 4.6 ng/ml per 10⁵ cells per 24 hr; such a cytokine displayed a specific biologic activity, as revealed by the stimulation of blast cell proliferation and the inhibition of lymphokine activated killer cell (LAK) induction by IL-2. After 200 Gy irradiation, IL-4 release remained detectable for 5 weeks, whereas cell proliferation ceased within 7 days. Morphology and immunophenotypic characteristics of the parental cell line (expression of MHC classes I and II, ICAM-1, LFA 3, melanoma-associated antigens, etc.) were retained by the IL-4 gene-transduced melanoma as assayed by microscopy and immunofluorescence; likewise, susceptibility to lysis by LAK cells as well as a T-cell clone recognizing the Melan-A/MART-1 antigen did not change. These results, together with the lack of replication-competent retrovirus, suggest that the Me14932/IL-4 cell line displays suitable characteristics for its use in the treatment of HLA-matched melanoma patients.

Gene therapy approaches have recently been investigated for the treatment of acquired immunodeficiency syndrome (AIDS), both in preclinical and clinical studies, because more traditional antiviral agents have proven to be of limited effectiveness. We have previously shown that long-term protection against both laboratory and clinical isolates of human immunodeficiency virus type 1 (HIV-1) was conferred by HIV-regulated diphtheria toxin A (DT-A) chain in a human T cell line. Because the monocyte/macrophage cell is an important reservoir for HIV-1 in infected individuals, we sought here to determine whether HIV-regulated DT-A would also be effective in the promonocytic cell line U937. We report here that long-term protection, conferred by HIV-regulated DT-A, was observed in U937 cells, but that protection was dependent on the stock of HIV IIIB used for challenge. HIV production was measured by p24 assays, polymerase chain reaction (PCR) for HIV vif, gag, and reverse transcriptase (RT) sequences, and cocultivation with peripheral blood mononuclear cells (PBMCs). Complete protection was seen in DT-A-transduced cells with a stock of IIIB propagated on H9 cells and titered on peripheral blood mononuclear cells (PBMCs), while protection in these same cells with a second stock of IIIB, propagated and titered on H9 cells, was only partial and dose dependent.

The particulate form of the major envelope or surface (S) protein of hepatitis B virus (HBV) can be taken up by antigen-presenting cells and processed for class I presentation as an exogenous protein. We have used several DNA plasmid vectors expressing the HBV envelope proteins to determine whether these sequences are able to induce cytotoxic T lymphocyte (CTL) responses in BALB/c mice after intramuscular DNA injection. A potent and specific induction was obtained, which can be detected ex vivo using either specific or nonspecific (interleukin-2) stimulation in cell culture, and the DNA-primed CTL responses are stronger than those obtained with protein injection with either stimulation protocol. The CTL response induced by DNA-based immunization is both canonical and highly specific as indicated by the nature of the epitope presented (amino acids 28–39), the class I allele used (L^d), and the T lymphocytes involved (CD8⁺). The CTL response is initiated between 3 and 6 days after DNA injection. By 6–12 days after a single DNA injection, ex vivo cytolytic activity is nearly maximal, and similar high levels of activity can still be detected 4 months after injection. The possibility is discussed that the unusual mode of delivery of the antigen to the immune system provided by in situ expression might allow HBV envelope antigen to be taken up and processed for class I presentation as an exogenous protein in addition to activating potentially the classical endogenous pathway.

In vivo gene transfer of angiogenic growth factors represents a potential approach to the treatment of ischemic diseases. The present study examined the in vitro and in vivo effects of two replication-deficient recombinant adenovirus (Ad) vectors coding for human acidic fibroblast growth factor (aFGF_1–154). One vector codes for the nonsecreted form of the peptide (AdCMV.aFGF_1–154), and the other vector codes for a recombinant, secreted form (AdCMV.sp+aFGF_1–154). AdCMV.NLSβgal, an adenovirus vector coding for β-galactosidase (β-Gal), was used as a control. Assessment of proliferation of starved human umbilical vein endothelial cells infected with AdCMV.aFGF_1–154 and AdCMV.sp+aFGF_1–154 (20 pfu/cell) showed approximately 6- and 10-fold increase in cell number over control, respectively. Infection with AdCMV.sp+aFGF_1–154 and with AdCMV.aFGF_1–154 enhanced endothelial cell differentiation into capillary-like structures in vitro. However, this effect was significantly more pronounced with AdCMV.sp+aFGF_1–154 than with AdCMV.aFGF_1–154. Angiogenesis in vivo was assessed by injecting subcutaneously into mice 750 μl of reconstituted basement membrane proteins (Matrigel) and the Ad vectors (2 × 10⁸ pfu). After 14 days, there was histologic evidence of neovascularization in the animal's tissue surrounding the Matrigel plugs with AdCMV.aFGF_1–154 and AdCMV.sp+aFGF_1–154. Further, the hemoglobin content of the Matrigel plugs with AdCMV.aFGF_1–154 and with AdCMV.sp+aFGF_1–154 was, respectively, 2.3- and 2.6-fold higher than with AdCMV.NLSβgal. Together, these observations support the concept that adenovirus vectors coding for various forms of acidic FGF_1–154 may be used to induce angiogenesis in vivo and may provide a new therapeutic approach to ischemic diseases.

Transfer of the herpes simplex virus type-1 thymidine kinase (HSV-tk) gene into tumor cells followed by ganciclovir (GCV) administration, will provide selective tumor cell killing. We studied the effect of herpes simplex virus thymidine kinase (HSV-tk) expression level on the HSV-tk/GCV-mediated “bystander effect.” Clones of HSV-tk-transduced rat glioma cells (9L) were isolated that stably expressed with different levels of HSV-tk. All clones studied had similar sensitivity to ganciclovir with IC₅₀ values ranging from 0.45 to 1.3 μM. Within certain enzyme level thresholds, in vitro evaluation of the bystander effect has shown that clones with higher level of HSV-tk expression exhibited a better bystander effect. Interestingly, the bystander effect was observed between different cell types. Both the transduction efficiency and bystander effect are essential factors for the success of the antitumor effect by the HSV-tk/prodrug GCV suicide gene system.

Duchenne muscular dystrophy (DMD) is a lethal genetic disorder for which there is currently no effective treatment. Although clinical application of adenoviral vector-mediated gene transfer has not been fully developed, it shows promise for the treatment of DMD. One significant problem posed by adenoviral vector-mediated gene transfer for DMD is that currently available adenoviral vectors cannot accommodate the entire 14-kb dystrophin cDNA. To address this problem, we selectively deleted regions of the murine dystrophin cDNA to produce truncated constructs. We created three constructs, each with an in-frame deletion of a segment (3.0, 4.4, and 5.7 kb) of the spectrin-like repeat region of dystrophin. As an additional modification, we removed the majority of the 3′ untranslated region of the cDNA in expression vectors encoding some of these truncated constructs. Comparative quantitative expression studies after transfection into COS and C2C12 mouse muscle cells demonstrate variations in the level of expression with different deletions in the spectrin-like repeat region. Furthermore, deletion of the 3′ untranslated region was tested for one recombinant construct and resulted in a reduction in the level of expression in both cell culture systems. Toward the ultimate goal of gene transfer therapy for DMD, we created an adenoviral vector from one of our truncated constructs. Using this vector, we demonstrated truncated dystrophin expression in vitro in primary mdx (dystrophin-deficient) muscle cells and in vivo in mdx mouse muscle. In vivo, recombinant dystrophin was properly localized to the muscle membrane.

Mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) manifest on the nasal epithelial surface of individuals with cystic fibrosis (CF) by Na⁺ hyperabsorption and diminished β-agonist-induced Cl¯ conductance, resulting in an abnormal bioelectric phenotype across the nasal epithelium. A clinical trial was conducted to determine if a replication-deficient, recombinant adenovirus vector containing a normal copy of the CFTR cDNA (AdCFTR) could, when administered to the nasal epithelium, correct the abnormal bioelectric phenotype. Nine individuals with CF received 2 × 10⁵ to 2 × 10^8.5 plaque forming units of AdCFTR to the epithelium of one nostril. Measurements made included: baseline electrical potential difference (PD) between the surface of the nasal epithelium and the interstitial fluid, change in PD in response to amiloride, which inhibits apical Na⁺ channels, and change in PD in response to isoproterenol in a low Cl¯ solution, a measure of cAMP-regulated Cl¯ conductance. The functional integrity of the epithelium was evaluated by the PD response to ATP. Each individual served as their own control with measurements made in the nostril to be treated before AdCFTR administration, and in the contralateral untreated nostril. On the average, in the treated nostril over 2 weeks after the local administration of the adenovirus vector compared to measurements made in the same nostril before treatment, baseline PD decreased toward normal (–53.3 ± 4.0 to –34.6 ± 3.4, p = 0.01), response to amiloride decreased toward normal (36.9 ± 4.7 to 19.7 ± 3.0, p = 0.02), and response to low CI¯ and isoproterenol increased toward normal (–4.5 ± 1.5 to –9.1 ± 2.1, p = 0.05). There were no changes in response to ATP (–15.3 ± 2.7 to –15.8 ± 1.9, p = 0.39), suggesting that the epithelium remained functionally intact. Importantly, there were no significant changes in measurements made in the untreated nostril. While limited to the nasal epithelium, these data suggest an adenovirus vector can safely deliver sufficient CFTR cDNA function to improve the abnormal CF bioelectric phenotype.

Lipofectin-mediated gene transfer was used to introduce plasmid harboring the tyrosine hydroxylase (TH) gene into the striatum of rats with lesions of the nigrostriatal pathway. The rotational asymmetry of Parkinson disease model rat was reduced quickly and significantly, suggesting that plasmid–DNA-transfected brain cells can generate l-dopa locally in the striatum in quantities sufficient to compensate partially for the loss of intrinsic striatal dopaminergic input. Immunohistochemical staining and reverse transcription polymerase chain reaction (RT-PCR) also confirm that striatal cells can express exogenous TH gene. Such in vivo plasmid DNA transfer strategy may be useful in other neurologic disease therapy, especially acute brain insults.