Human Gene Therapy

The T cell co-stimulatory molecule B7-1 was transduced into a poorly immunogenic murine neuroblastoma cell line (Neuro-2a, N-2a) alone or in combination with MHC class II genes to test the ability of these genes to stimulate antitumor immunity. N-2a cells transduced with B7-1 exhibited reduced tumorigenicity, whereas N-2a cells overexpressing both MHC class II (syngeneic, I-A^k) and B7-1 totally abrogated tumorigenicity. Rejection of I-A^k/B7-1 cells was dependent on both CD4⁺ and CD8⁺ T cells. The ability of both vaccines to induce protection against parental N-2a was temporally dependent on the time of secondary N-2a challenge. To investigate the immunity generated by N-2a/B7-1 and N-2a/I-A^k/B7-1 vaccines, we tested the ability of these modified cells to stimulate in vitro the proliferation of syngeneic splenocytes from naive mice. A significant increase in splenocyte proliferation was observed with N-2a/I-A^k/B7-1 cells compared to N-2a cells. We also determined that vaccination with N-2a/I-A^k/B7-1 cells was able to generate cytotoxic T cell responses to unmodified N-2a cells. The introduction of B7-1 and I-A^k into N-2a was able to convert a poorly immunogenic tumor to a highly immunogenic one; however, mice bearing large established unmodified tumors had little response to vaccination with N-2a/I-A^k/B7-1 cells. Our results emphasize the importance of tumor immunogenicity in the treatment of established tumors with MHC class II/B7-1 tumor cell vaccines.

Previous studies have shown that dihydrofolate reductase (DHFR) gene transfer protects marrow from methotrexate (MTX)-mediated toxicity; however, MTX treatment in vivo has not convincingly been shown to enrich DHFR-transduced progenitors or stem cells. Experiments were performed to better characterize the hematological effects of MTX, and maneuvers were tested with the aim of improving the utility of MTX as an agent for in vivo selection. Progenitors were assayed as colony forming unit cells in culture (CFU-C) and in the spleens of irradiated mice (day 11 CFU-S). A single injection of MTX at doses up to 250 mg/kg (more than three times the LD₁₀) failed to reduce CFU-C numbers significantly in the femur or spleen assayed 1–3 days later. However, consistent declines in the number of mononuclear cells per femur reflected a significant depletion of nonclonogenic precursor cells. Preceding administration of pegylated stem cell factor (SCF), 100 μg/kg per day, increased CFU-C killing by a single dose of 5-fluorouracil (5-FU) 15- to 65-fold in the femur, and 5- to 15-fold in the spleen, consistent with previous reports. In contrast, despite preceding SCF administration there was no significant progenitor killing by MTX. Similar results were obtained using a second folate analog, trimetrexate. These results suggest that the mechanism by which folate analogs exert their hematological toxicity is through the depletion of relatively mature, nonclonogenic precursor cells, and not by killing progenitors. This information is relevant to the use of DHFR in gene therapy protocols, and suggests that folate analogs are poorly suited agents for selection at the level of clonogenic progenitor cells in vivo.

Adenovirus and adeno-associated virus (AAV) are eukaryotic DNA viruses being developed as vectors for human gene therapy. The strengths of each system have been exploited in a novel vector that is based on an ade novirus–AAV hybrid virus incorporated into a plasmid-based molecular conjugate. Efficient rescue and replication of the recombinant AAV genome in this hybrid required transient expression of rep. This feature was incorporated into the transducing particle by conjugating a rep expression plasmid to the hybrid virus through a polylysine bridge. The resulting particle is an attractive vehicle for gene therapy because it is easily manufactured and capable of efficiently transducing cells with the end result being rescue and replication of the recombinant AAV genome. This particle is also useful in the production of recombinant AAV resulting in yields 10-fold greater than that achieved with transfection-based protocols.

Long-term marrow cultures (LTMCs) were established from 27 human marrows. Hematopoietic cells were subjected to multiple rounds of exposure to retroviral vectors during 3 weeks of culture. Seven different retroviral vectors were evaluated. LTMCs were assessed for viability, replication-competent retrovirus, progenitors capable of proliferating in immune-deficient mice, and gene transfer. The average number of adherent cells and committed granulocyte-macrophage progenitors (CFU-GM) recovered from LTMCs was 28% and 11% of the input totals, respectively. There was no evidence by marker rescue assay or polymerase chain reaction (PCR) of replication-competent virus production during LTMC. No toxicity to cellular proliferation due to the transduction procedure was observed. The adherent layers of LTMCs exposed to retroviral vectors were positive for proviral DNA by PCR and by Southern blot analysis. Fifty-three percent of 1,427 individual CFU-GM from transduced LTMC adherent layers were positive for vector-derived DNA. For neo^r-containing vectors, the average G418 resistance was 28% of 1,393 LTMC-derived CFU-GM. Forty percent of 187 tissues from 30 immune-deficient mice injected with human LTMC cells were positive for human DNA 4–5 weeks after adoptive transfer. These studies indicate that multiple exposures of human LTMCs to retroviral vectors result in consistent and reproducible LTMC viability and gene transfer into committed progenitors. Our results further support the use of transduced LTMC cells in clinical trials of hematopoietic stem cell gene transfer.

Recombinant adeno-associated viruses (AAV) are among the most promising vectors for gene therapy of genetic diseases, including cystic fibrosis (CF). However, because of its small genome size, the capacity of AAV to package a therapeutic gene is limited. The efficiency of packaging the cystic fibrosis transmembrane conductance Regulator (CFTR) gene into AAV will be an important factor in determining whether recombinant AAV can be developed as a vector for transferring CFTR cDNA to the airway epithelia of patients with CF. Current understanding of the AAV biology suggests that AAV can package a genome slightly larger than the size of a wild-type genome. The precise range of the genome size and the efficiency of packaging have not been defined. Using a series of AAV vectors with progressively increasing genome size, we were able to analyze quantitatively the packaging efficiency in relation to the vector size and to determine the size limit for packaging. The packaging efficiencies of AAV vectors of variable sizes were determined directly by assaying DNA contents of viral particles, and indirectly by analyzing their efficiency in transfer of a chloramphenicol acetyltransferase (CAT) reporter gene into target cells. Our studies showed that the optimal size of AAV vector is between 4.1 and 4.9 kb. Although AAV can package a vector larger than its genome size, up to 5.2 kb, the packaging efficiencies in this large size range were sharply reduced. When the AAV genome size was smaller than 4.1 kb, the packaging efficiency was also suboptimal. In contrast, when the size of the genome was less than half the length of the wild-type genome, two copies of the vector were packaged into each virion, suggesting that the copy number control during packaging is a “head-full” mechanism. Because the length of the minimal cDNA of CFTR is about 4.5 kb, these results suggest it is possible to package the CFTR gene into AAV if the combined length of transcriptional elements and ITRs is kept under 500 bp. The results of this study are important for directing the design of AAV vectors for efficient gene transfer, as well as for a better understanding of the mechanism of AAV genome packaging.

Hepatic gene therapy might correct the clinical manifestations of several genetic disorders in patients. Although retroviral vectors with a strong liver-specific promoter can result in stable and therapeutic levels of expression of genes from the liver, application of these techniques in humans is limited by the need to perform one or more invasive procedures to achieve ex vivo or in vivo transduction of hepatocytes. In vivo delivery involves injection of retrovirus into the portal vein during liver regeneration. Although transduction is efficient and specific for the liver, induction of hepatocyte replication requires a 70% partial hepatectomy or administration of a liver toxin. An alternative method for inducing hepatocyte replication is to occlude branches of the portal vein. This results in apoptosis of hepatocytes in the occluded lobes and compensatory replication of the hepatocytes in the nonoccluded lobes. We demonstrate here that portal branch occlusion is nearly as effective as partial hepatectomy at facilitating retroviral vector transduction in vivo and has a lower morbidity. Portal branch occlusion could be performed in larger animals by minimally invasive techniques and has been used safely to treat human patients with liver cancer. Portal branch occlusion might ultimately be used in humans to facilitate retroviral vector transduction in vivo for the treatment of genetic diseases.

Cationic polymers can self-assemble with DNA to form polyelectrolyte complexes capable of gene delivery, although biocompatibility of the complexes is generally limited. Here we have used A-B type cationic-hydrophilic block co-polymers to introduce a protective surface hydrophilic shielding following oriented self-assembly with DNA. Block co-polymers of poly(ethylene glycol)–poly-l-lysine (pEG-pLL) and poly-N-(2-hydroxypropyl)methacrylamide–poly(trimethylammonioethyl methacrylate chloride) (pHPMA–pTMAEM) both show spontaneous formation of complexes with DNA. Surface charge measured by zeta potential is decreased compared with equivalent polycation–DNA complexes in each case. Atomic force microscopy shows that pHPMA–pTMAEM/DNA complexes are discrete spheres similar to those formed between DNA and simple polycations, whereas pEG–pLL/DNA complexes adopt an extended structure. Biological properties depend on the charge ratio of formation. At optimal charge ratio, pEG–pLL/DNA complexes show efficient transfection of 293 cells in vitro, while pHPMA–pTMAEM/DNA complexes are more inert. Both block co-polymer-DNA complexes show only limited cytotoxicity. Careful selection of block co-polymer structure can influence the physicochemical and biological properties of the complexes and should permit design of materials for specific applications, including targeted delivery of genes in vivo.

Neurotrophic factors hold promise for the treatment of neurodegenerative diseases. Intrathecal transplantation of polymer encapsulated cell lines genetically engineered to release neurotrophic factors provides a means to deliver them continuously behind the blood–brain barrier. Long-term delivery, however, may benefit from the use of conditionally mitotic cells to avoid the overgrowth observed with continuously dividing cell lines. Myoblast lines have all the advantages of dividing cell lines, i.e., unlimited availability, possibility for in vitro screening for the presence of pathogens, suitability for stable gene transfer and clonal selection. Furthermore they can be differentiated into a nonmitotic stage upon exposure to low-serum-containing medium. In this study, mouse C₂C₁₂ myoblasts were transfected with a pNUT expression vector containing the human ciliary neurotrophic factor (CNTF) gene. hCNTF expression and bioactivity were demonstrated by Northern blot, ELISA assay, and measurement of choline acetyltransferase (ChAT) activity in embryonic spinal cord motor neuron cultures. One C₂C₁₂ clone was found to secrete 200 ng of CNTF/10⁶ cells per day. The rate of secretion of hCNTF was not altered upon differentiation of C₂C₁₂ myoblasts. A bromodeoxyuridine (BrdU) proliferation assay indicated that approximately 12% of the myoblasts continue to divide after 4 days in low-serum-containing medium. The presence of the herpes simplex thymidine kinase gene (HSV-tk) in the expression vector, however, provides a way to eliminate these dividing myoblasts upon exposure to ganciclovir, therefore increasing the safety of the encapsulation technology using established cell lines. Encapsulated hCNTF-C₂C₁₂ cells can partially rescue motor neurons from axotomy-induced cell death. In adult rats, intrathecal implantation of encapsulated hCNTF-C₂C₁₂ cells or control C₂C₁₂ confirmed the long-term survival of these cells and their potential use as a source of neurotophic factors for the treatment of neurodegenerative diseases.

Although long-term expression of therapeutic molecules is necessary for the treatment of permanent deficiencies, short-term expression of therapeutic molecules inducing local or systemic effects is preferable in clinical situations where temporary substitution is the goal. One such clinical setting is the administration of hematopoietic growth factors in cancer chemotherapy-induced myelosuppression. Several plasmid vectors containing the human granulocyte colony-stimulating factor (G-CSF) gene under transcriptional control of different regulatory elements were constructed. In vitro production of G-CSF by nonvirally transfected murine fibroblast clones initially increased after lethal irradiation and was detectable for at least 12 days. We also demonstrate that a single injection of irradiated G-CSF-secreting fibroblasts leads to accelerated hematopoietic recovery and mobilization of committed peripheral blood progenitor cells equivalent to that achieved by twice daily s.c. administration of high doses of recombinant human G-CSF. Using dicistronic vectors, high levels of G-CSF secretion were also obtained in human fibroblasts.

Five different single-chain antibody fragments (scFv) against human cell-surface antigens were displayed on murine ecotropic retroviral vectors by fusing them to the Moloney SU envelope glycoprotein. The spacing between the scFv and the SU glycoprotein was varied by fusing the scFv to residue +7 or to residue +1 of Moloney SU and by inserting linker sequences of different lengths between the domains. All of the chimeric envelopes were efficiently incorporated into vector particles and could bind to human cells through their displayed antibody fragments, but did not infect them. The spacing between the scFvs and the SU glycoproteins had no significant effect on the efficiency of envelope expression or viral incorporation and did not affect the binding properties of the chimeric envelopes, nor did it influence the efficiency of targeted gene delivery to human cells by scFv-displaying vectors. However, on murine fibroblasts the infectivity of vectors incorporating the chimeric envelopes was strongly influenced by the length of the interdomain spacer. The titers were very low when the single-chain antibodies were fused through a tripeptide linker to SU residue +7 and were greatly enhanced (up to 10⁵-fold) when they were fused to SU residue +1 through a heptapeptide linker. These results point to the importance of steric interactions between the domains of chimeric envelope glycoproteins and may have implications for retroviral vector design for human gene therapy.

Retrovirus-mediated gene transfer is currently the most common method for the application of genetic therapy to cancer and many inherited and acquired disorders. Here we report the generation of an amphotropic producer cell line (CA2) that synthesizes viral particles carrying a bicistronic cassette in which the selectable MDR1 cDNA encoding P-glycoprotein (P-gp) a multidrug efflux pump, and the human glucocerebrosidase (GC) gene are transcriptionally fused. Transduction of human Gaucher fibroblasts with this recombinant virus allowed coordinate expression of P-gp and GC. Treatment of the transduced fibroblasts with various cytotoxic substrates of P-gp selected for cells with increased expression of GC, which paralleled the stringency of drug selection. Thus, selection of the genetically modified Gaucher fibroblasts in 1 μg/ml colchicine raised their GC activity levels from nearly undetectable to those present in WI-38 normal human fibroblasts, correcting the enzyme deficiency present in Gaucher cells. Moreover, by simultaneously inhibiting the P-gp pump, it was possible to use much lower concentrations of colchicine to select for high-level expression of MDR1 and GC. Thus, selection with colchicine at 5 ng/ml in combination with the P-gp inhibitors verapamil or PSC 833 produced a complete correction of the GC deficiency in the CA2-transduced fibroblasts. These combination regimens, already in clinical use for the treatment of multidrug-resistant malignancies, may prove useful in gene therapy trials when utilized for high level selection of a nonselectable gene such as glucocerebrosidase when transcriptionally fused to the MDR1 gene.

The aim of this study was to assess the feasibility of using gene transfer to salivary glands to direct the systemic delivery of therapeutic protiens in vivo. We used a replication-deficient recombinant adenovirus vector (Adα1AT) that encodes human α1-antitrypsin (hα1-AT), which we used as a marker protein. Adα1AT (5 × 10⁹ pfu) was administered by retrograde ductal instillation to the submandibular glands of male rats. The amount of hα1-AT found in the salivary glands, saliva, serum, and other tissues was analyzed by a sensitive enzyme-linked immunosorbent assay (ELISA). Maximal levels of the marker protein were detected at 24–48 hr post-virus administration for glands (274 ng/mg protein), saliva (∼313 ng/ml), and serum (∼5 ng/ml) Serum levels remained elevated for 96 hr, whereas the measured half-life for the marker protein was ∼2 hr Generally little to no hα1-AT was detectable in most other organs. However, we were able to measure low levels of marker protein in tissues immediately surrounding infected glands. In all animals studied, levels of hα1-AT were higher in the glandular venous effluent than in arterial blood. Similar results were found with parotid glands. The aggregate data demonstrate that salivary glands may be a target for the nonsurgical, systemic delivery of transgene-encoded therapeutic proteins for diseases that require relatively low circulating protein levels.

The increasing use of nucleic acid-based therapeutics has created a need for new methods of determining tissue distribution and levels. Radiolabel methods may not always be appropriate because nucleic acids are easily degraded. Quantitation using the polymerase chain reaction (PCR) has the advantage that only continuous stretches of DNA will be amplified. In situ hybridization allows detection of specific sequences in histological preparations. We have used quantitative PCR and in situ hybridization techniques to study the pharmacokinetics and distribution of PGagPol (a potential anti-HTV plasmid vaccine) in rabbits. Samples were obtained 4 hr, 24 hr, 7 days, and 28 days after intramuscular injection of 100 μg or 400 μg of plasmid. A simplified procedure for collecting and processing tissues for PCR that minimizes the risk of contamination was developed. Using PCR, plasmid was found principally in the skin and muscle of the injection site and in blood plasma. At 4 hr after dosing with 400 μg, the plasmid was detected at the injection site with mean copy numbers of 10⁶ (in muscle) and 4 × 10⁴ (in skin) per microgram of tissue. Plasmid copy number declined rapidly in muscle during the first 24 hr and was undetectable at 7 and 28 days after injection. The decline was slower in the skin, and the plasmid was still detectable at 28 days. With in situ hybridization, plasmid was detected in muscle, mainly in the perimysium and to a lesser degree in the endomysium and within the muscle fibers. These data indicate that quantitative PCR and in situ hybridization are sensitive methods for examining tissue distribution of DNA used for gene therapy.

Genetically modified murine skeletal myoblasts were tissue engineered in vitro into organ-like structures (organoids) containing only postmitotic myofibers secreting pharmacological levels of recombinant human growth hormone (rhGH). Subcutaneous organoid implantation under tension led to the rapid and stable appearance of physiological sera levels of rhGH for up to 12 weeks, whereas surgical removal led to its rapid disappearance. Reversible delivery of bioactive compounds from postmitotic cells in tissue engineered organs has several advantages over other forms of muscle gene therapy.