STRATEGIES IN GENE THERAPY GENE THERAPY FOR CANCER Gene Therapy for Primary and Metastatic Pancreatic Cancer GENE THERAPY FOR PULMONARY DISEASES Gene therapy for acquired diseases Gene therapy for Acute Lung Diseases Gene therapy for Inherited Lung diseases   Gene therapy can either be applied ex vivo or in vivo. Ex vivo methods are technically simpler with regard to vector transfer & gene expression, but surgery is required to obtain & replace the cells. To enhance in vivo delivery the target organ may be stimulated, for example a partial hepatectomy will improve retroviral transduction to the liver , however minimal manipulation of the patient would be desirable. For some diseases the pathology affects the function of a particular organ which must be directly treated, e.g. cystic fibrosis in the lungs, inflammatory bowel disease , Parkinsons disease in the corpus striatum region of the brain & retinitis pigmentosa . Other diseases are systemic in their effects, e.g. haemophilia & metabolic diseases such as adenosine deaminase deficiency. For these diseases the common sites for therapy are the liver, gut & muscle . These sites are chosen for their ease of access, bulk & metabolic activity. Monogenic recessive diseases only require the functional gene to be expressed, often therapeutically useful levels being much lower than that those found in normal individuals. Monogenic dominant diseases require that the aberrant gene is silenced, usually by means of an anti-sense DNA which is complementary to the aberrant gene. GENE THERAPY FOR CANCER Many gene therapy protocols to date have concentrated upon treatments for cancer. Though many cancers have a genetic predisposition, they all involve acquired mutations, & as they progress their cells become less differentiated & more heterogeneous with respect to the mutations they carry. In general cancers have at least one mutation to a protooncogene (yielding an oncogene) & at least one to a tumour suppressor gene, allowing the cancer to proliferate. The range of different cancers encountered & the mutations they carry, have led to a variety of strategies for gene therapy namely; immunopotentiation, oncogene inactivation, tumour suppressor gene replacement, molecular chemotherapy & drug resistance genes. The aim of immunopotentiation is to enhance the response of the immune system to cancers, thereby leading to their destruction. Passive immunotherapy aims to increase the pre-existing immune response to the cancer whilst active immunotherapy initiates an immune response against an unrecognised or poorly antigenic tumour. Passive immunotherapy usually involves harvesting tumour infiltrating lymphocytes & treating them to express increased cytokines e.g. IL-2 & TNF-alpha. The cell population is then expanded in vitro & returned to the patient. Tumour cells are used for active immunotherapy, genetically modifying them to increase expression of antigen presenting molecules/costimulatory molecules, local concentrations of cytokines (e.g. IL-2; Lemig et al, 1996) or tumour antigens (erbB2 oncoprotein; Disis et al, 1994). The cells are then irradiated prior to being returned to the patient, preventing the reintroduction of replication competent tumour cells. These approaches have been termed cancer vaccines. Oncogene inactivation uses the same techniques employed for dominantly inherited monogenic diseases. The oncogene may be targeted at the level of the DNA, RNA transcription or protein product. Oligodeoxynucleotides are short single stranded pyridimine rich DNA sequences that form a triple helix with purine rich double stranded DNA sequence. Oligodeoxynucleotides are designed in a sequence specific manner to target the promoter regions of oncogenes.At the RNA level antisene techniques prevent transport & translation of the oncogene mRNA by providing a complementary RNA molecule (e.g. c-myc; Collins et al, 1992). Ribozymes, antisense oligoribonucleotides with a cleavage action, will also reduced the stability of oncogene mRNA. Transport of the oncogene product to the cell surface can be prevented by a single chain antibody with specificity for the oncogene product & a localisation signal for the endoplasmic reticulum.. Despite multiple genetic abnormalities, restoration of the tumour suppressor gene, such as p53, can be sufficient to cause cellular apoptosis & arrest tumour growth. Moreover, expression of p53 is synergistic with chemotherapeutic drugs such as cisplastin & adjacent tumour cells that have not been transduced are killed, in what is termed the bystander effect . The p53 gene has been identified as important in a range of cancers & though less elegant than oncogene inactivation techniques, this method has proved robust enough to be included in a number of clinical trials. Other tumour suppressor genes may also prove useful e.g. BRCA1sv. An alternative means of killing a tumour cell is to transduce a gene coding for a toxic product, known as molecular chemotherapy. The gene of choice is usually herpes simplex virus thymidine kinase (HSV/TK) which converts the prodrug ganciclovir into toxic metabolites. The effected cell is supported via the gap junctions of adjacent cells, until the toxin burden is too great killing both the affected cell & its neighbours. An advantage to this system is that all the transduced cells will be killed, allowing allogenic tumour cells to be prepared in advance , Oldfield & Ram, 1995). HSV/TK is also used in other gene therapy protocols, allowing the treatment to be aborted at any time. Many cancer chemotherapy regimes are limited by their low therapeutic index, as determined by heamopoetic cells. By harvesting precursor bone marrow cells & transducing them with the gene MDR1, coding for the drug efflux protein p170, a population of resistant cells can be cultured. When returned to the patient, much higher doses of chemotherapy may be used. CLINICAL TRIALS Numerous systems have been demonstrated in vitro & with animal models. Phase 1 human trials are primarily designed to demonstrate safety & efficacy & will not provide complete cures. The patients chosen have advanced metastatic cancer & the early results are comparable to single agent chemotherapy. Clinically, viral gene transfer to cancer cells has proved more efficient than expected from normal organ gene transfer & viral vectors can spread through three dimensional cell matrices . Perhaps due to the heterogeneous nature of many malignant tumours, the most successful approaches do not rely on manipulating the immune system, rather act as adjuncts to conventional therapies (drug resistance genes) or use established agents (molecular chemotherapy). The morbidity associated with cancer gene therapies is significantly lower than of conventional treatments, therefore more aggressive application of gene therapies may be beneficial. Development of therapies for non malignant diseases have been hampered by the short duration of transgene expression. Recent advances however, make it likely that monogenic recessive diseases, such as haemophilia, will be treatable by administering a single dose of the gene vector Current objectives include improvement of cell targeting through vector & promoter specificity (discussed above) & reducing the immune response to the current vectors. As the understanding of the molecular biology, immunology & virology underlying vector technology & oncology improves, so will our ability to treat cancer. Many of the developments reviewed above are present only in isolated systems, therefore consolidation of existing technology may yield significant improvements. Public expectation, though understandable & used by scientists to generate funds, should be tempered as gene therapy will not be the cure all in the near future. However, in conjunction with existing therapies, gene therapy will substantially reduced the burden of morbidity experienced by an ageing population. Gene Therapy for Primary and Metastatic Pancreatic Cancer With Intraperitoneal Retroviral Vector Bearing the Wild-Type p53 Gene Metastatic pancreatic cancer is uniformly fatal because no effective chemotherapy is available. Mutations in the p53 tumor suppressor gene are found in up to 70% of pancreatic adenocarcinomas. We examined the efficacy of a retroviral vector containing the wild-type p53 gene on metastatic pancreatic cancer in a nude mouse model. METHODS Bxpc3 human pancreatic cancer cells were transduced with either a retroviral p53 vector or an LXSN empty vector. Cells were examined for incorporation of tritiated thymidine to determine the effect of p53 retroviral transduction on DNA synthesis, and a TACS2 assay for apoptosis was performed. The functional activity of p53 in transduced cells was assessed by Western blot analysis with an antibody to WAF1/p21. In vivo effects of intraperitoneal injections of the p53 vector were examined in a nude mouse model of peritoneal carcinomatosis. RESULTS Cells treated with the p53 vector exhibited a 59% to 85.5% reduction in cell number compared with the control cells (P < .05). p53-treated cells demonstrated decreased incorporation of tritiated thymidine (12.7% ± 0.7% vs 17.5% ± 1.4%; P = .002), increased staining for apoptosis, and increased expression of the WAF1/p21 protein. Treatment of nude mice with the retroviral p53 vector resulted in a significant inhibition of growth of the primary pancreatic tumor, as well as the peritoneal tumor deposits, compared with the LXSN control vector. CONCLUSIONS: Intraperitoneal delivery of a retroviral p53 vector may provide a novel treatment approach for peritoneal carcinomatosis from pancreatic cancer. Intercellular Communication Mediates the Bystander Effect During Herpes Simplex Thymidine Kinase/Ganciclovir-Based Gene Therapy of Human Gastrointestinal Tumor Cells A bystander effect is described when nontransduced or genetically unmodified cells are killed during death of genetically modified tumor cells transduced with a suicide gene. The "bystander effect" greatly enhances the efficacy of the herpes simplex virus-thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy approach for cancer. The mechanism of the bystander effect is controversial. In this study, we examined the role of intercellular gap junction communication (GJIC) for the bystander effect in human gastrointestinal tumor cells. Our results show that the extent of the bystander effect varied amongst the tumor cell lines; pancreatic cancer cells BXPC-3 exhibited excellent bystander effects in vitro and in vivo studies whereas other gastrointestinal tumor cell lines such as pancreatic cancer cells MIAPACA-2, and colon cancer cells HT-29 showed poor bystander effects. Bystander effects were only found in the presence of cell-to-cell contact. The extent of the bystander effect was independent of the level of HSV-TK activity in the transduced tumor cells and was correlated with GJIC as demonstrated by an in vitro dye-transfer assay. Expression of the mRNA levels of gap junction protein connexin 43 was 8- to 26-fold or greater and connexin 26 gene expression was 2- to 229-fold greater in BXPC-3 cells compared to HT-29, MIAPACA-2, and PANC3 cells. Our results suggest that intercellular communication is essential for the bystander effect. The correlation between GJIC and the extent of the bystander effect suggest a role for GJIC in mediating the bystander effect. Analysis of tumors for GJIC or expression of gap junction proteins may identify the subset of patients suitable for gene therapy with the HSV-TK/GCV approach. An Apoptosis-Inducing Gene Therapy for Pancreatic Cancer With a Combination of 55-kDa Tumor Necrosis Factor (TNF) Receptor Gene Transfection and Mutein TNF Administration Intratumoral injection of recombinant human tumor necrosis factor (TNF) for inoperable pancreatic cancer has shown some efficacy in suppressing tumor growth or decreasing tumor markers. However, complete regression has not yet been achieved, possibly due to a lack of TNF receptors on tumor cells or an abundance of intracellular resistance factors. Recently, two distinct types of TNF receptors, R55 and R75, were identified, which are responsible for signaling of cytotoxicity and of proinflammation, respectively. In this study, a novel type of suicide gene therapy is proposed that is based on transfection of the R55 gene into human pancreatic cancer cells (AsPC-1 and PANC-1) and subsequent administration of TNF. The transfectants from both cell lines showed higher TNF susceptibility than their parental cells. In vivo tumor formation of an AsPC-1 clone (clone 10) inoculated in nude mice was substantially suppressed by administration of TNF. For practical use of this strategy, however, the adverse effects of TNF may become an obstacle. We previously produced mutein TNF 471, which had a higher affinity for R55, superior antitumor activity, and fewer adverse effects. This mutein TNF 471 manifested greater antitumor activity against clone 10. Because the R55 receptor is known to be involved in augmentation of cellular immunity by TNF, mutein TNF 471 is also expected to be highly potent in this function. In fact, the mutein TNF 471 induced higher splenic natural killer cell activity in nude mice inoculated with clone 10 than did native TNF. This property of augumenting cellular responses may be advantageous in the eradication of viable tumor cells left untransfected in practical gene therapy regimens in which 100% transfection of the R55 gene into tumors is not feasible. Thus, gene therapy combining transfection of the TNF-R55 gene with administration of mutein TNF 471 may provide a new modality for the treatment of pancreatic cancer. In Vivo Adenovirus-Mediated Prodrug Gene Therapy for Carcinoembryonic Antigen-Producing Pancreatic Cancer In gene therapy for malignancy, the herpes simplex virus thymidine kinase (HSVtk)-ganciclovir (GCV) system has been widely used. For pancreatic cancer targeting, we estimated the therapeutic efficacy of gene transduction by an adenovirus-carrying HSVtk gene under the control of a carcinoembryonic antigen (CEA) promoter (AdCEAtk) followed by systemic administration of GCV. Four cell lines, CEA-producing Su.86.86. BxPC-3 (pancreatic cancer cells), MKN45 (gastric cancer cells) and CEA-nonproducing HeLa, were used for analysis of GCV sensitivity induced by adenoviral gene transduction. To evaluate the therapeutic efficacy of AdCEAtk and GCV administration in human CEA-positive pancreatic cancer in vivo, a subcutaneously implanted tumor-bearing nude mouse model was used. When the HSVtk gene was transduced with a ubiquitous promoter into these cells, increase of the GCV sensitivity was independent of CEA-production. In contrast, when the cells were transduced with a CEA promoter, the cell-killing effect of GCV was increased in only CEA-producing cells. For in vivo analysis, AdCEAtk was delivered into subcutaneously established tumors of Su.86.86 cells. Immunohistochemical staining of the tumor showed that HSVtk protein was expressed only in tumor cells, and tumor growth was markedly suppressed by administration of GCV. These results suggest that the adenovirus-mediated transfer of HSVtk gene with CEA promoter specifically increases the GCV sensitivity of CEA-producing pancreatic cancer cells in vitro and in vivo. This strategy may provide a useful tool for treating pancreatic cancer, especially CEA-producing tumor cells. Gene Therapy for Peritoneal Dissemination of Pancreatic Cancer by Liposome-Mediated Transfer of Herpes Simplex Virus Thymidine Kinase Gene Peritoneal dissemination is one of the most common complications of the malignancies of the digestive system, such as gastric or pancreatic cancers. Yet, no effective therapy has been established so far to alleviate this devastating and often fatal end-stage condition. Here we describe a novel approach of intraperitoneal (i.p.) lipofection of a suicidal gene to the pancreatic cancer cells in a mouse peritoneal dissemination model. A human pancreatic cancer cell line, PSN-1, was inoculated into the peritoneal cavity of nude mice. Eight days later, a herpes simplex virus thymidine kinase (HSV-TK) gene expression plasmid under a potent hybrid promoter CAG was injected as a DNA-lipopolyamine complex. Ganciclovir (GCV) was then administered for 8 days, and the mice were examined for tumor development at the 24th day after the tumor inoculation. Although all 24 control mice showed macroscopic peritoneal dissemination and solid tumors on the pancreas, 8 of the 14 mice treated with HSV-TK and GCV were free of tumors, and only a few small tumors were observed in the remaining 6 mice. Treatment-related toxicity was not observed. The semiquantitative reverse transcription polymerase chain reaction (RT-PCR) analysis suggested that the HSV-TK transgene was expressed in about 10% of tumor cells but not in the normal pancreas or in the small intestine. When the lacZ gene was transduced in place of the HSV-TK gene, the blue-stained cells were identified only in tumor nodules and not in normal organs. This preclinical study suggests the therapeutic feasibility of the i.p. lipofection-based suicidal gene/prodrug strategy for peritoneal dissemination of pancreatic cancer. Expression of Fas-estrogen Receptor Fusion Protein Induces Cell Death in Pancreatic Cancer Cell Lines Recently, a novel system to induce apoptosis was reported. Fusion protein between Fas and the ligand-binding domain of the estrogen receptor (MfasER) could cause apoptotic cell death in an estrogen-dependent manner on murine fibrosarcoma L929 cells and human cervical carcinoma HeLa cells. To investigate the application of this system to the gene therapy of pancreatic cancer, we introduced MfasER cDNA to six cell lines. Transiently expressed MfasER could cause cell death in all the cell lines tested. Furthermore, stably MfasER-expressing cells showed DNA fragmentation as early as 2 h and completely died in 48 h in the presence of estrogen. Combined with effective methods to introduce genes to pancreatic cancer selectively, MfasER would be a good tool for the gene therapy of pancreatic cancer in the future. Suppression of Pancreatic Cancer by the Dominant Negative ras Mutant, N116Y N116Y, H-ras mutant, possesses dominant negative activity to ras function. The aim of this study is to assess whether N116Y can inhibit the proliferation of pancreatic cancer cell lines carrying K-ras mutations and cause reversion of the malignant phenotype. We transfected an expression vector of N116Y, pZIP-N116Y, into eight human pancreatic cancer cell lines with K-ras mutations (PCI 10, 19, 24, 35, 43, 55, 64, and 66) by using a lipofection procedure. The growth inhibition activity of N116Y was evaluated by the colony-forming efficiency in selection medium. In order to examine the effect of N116Y on the neoplastic phenotype, we established N116Y-expressing clones and analyzed their growth ability in soft agar and tumorigenicity in nude mice. The growth of the eight pancreatic cancer cell lines was strongly inhibited by the transfection of pZIP-N116Y. Moreover, the N116Y-expressing clones became less spread and lost their anchorage-independent growth ability. Furthermore, they were nontumorigenic in vivo. N116Y significantly inhibits the growth of pancreatic cancer cell lines and causes reversion of the malignant phenotypes. These results suggest that N116Y may be a candidate gene for use in the gene therapy of pancreatic cancer. Back to Top GENE THERAPY FOR PULMONARY DISEASES Gene Therapy for Aquired Diseases Gene Therapy for Acute Lung Disease Gene Therapy for Inherited Lung Disease GENE THERAPY FOR ACQUIRED DISEASES The two key concepts presented here are: The controlled expression of specific genes can be viewed as using genes as drugs for acquired disease. To be successful in this application a thorough understanding of the molecular basis of disease is essential. There are three broad strategies for using genes as drugs: Hyperexpress a normal protective gene Express a normal but down-regulated gene Block expression of a foreign gene Examples of pulmonary diseases where each one of these strategies shows promise are as follows. Hyperexpress a Normal Protective Gene: Human -1 Antitrypsin (AAT) The AAT gene is expressed in the liver and the resultant protein is taken up by respiratory epithelial cells and secreted into the epithelial lining fluid. AAT protects the lung against injury from a variety of proteases. There are circumstances where you might want to have the lung cells themselves expressing the gene in order to offer more protection. For example, in Adult Respiratory Distress Syndrome (ARDS) AAT may be overwhelmed by the acute lung injury and hyperexpressing the gene could restore the normal balance between proteases and antiproteases. In HIV, a viral protease is responsible for cleaving the viral polypeptides into individual viral proteins which are then packaged into infectious virions. Inhibiting this process with intracellular production of AAT could theoretically prevent the assembly of infectious particles. Finally, respiratory syncytial virus (RSV) may require activation of a key viral protein with cellular proteases before it can be infectious. Thus, in these three situations, a short duration of hyperexpression of AAT at the site of the disease could terminate the pathologic process. Express a normal but down-regulated gene Idiopathic pulmonary fibrosis (IPF) in humans is a chronic disease characterized by pulmonary inflammation and fibrosis. The prognosis is dismal and no treatment is available other than lung transplant. Recently, lung fibroblasts from patients with IPF were shown to have reduced cyclooxygenase activity (COX) and thus were unable to generate the COX product, prostaglandin E2 (PGE2. Since PGE2 inhibits fibroblast proliferation, collagen synthesis, and cytokine release, it is postulated that this deficiency in generation of PGE2 is responsible for lung fibrosis. The sequence of events may be as follows: acute lung insult ---> deficient COX expression --->deficient generation of PGE2 --->lung fibrosis. If this is true, then pulmonary expression of COX should restore normal fibroblast function. Block expression of a foreign gene: antisense therapy for viral infections Antisense oligonucleotides are defined as complementary RNA or DNA oligonucleotides. By sequence-specific base pairing with the target DNA or RNA, there is inhibition of protein synthesis. The advantage of this approach is the exquisite sensitivity. It is estimated that an oligonucleotide of 15 to 17 bases will be specific for only one site in the entire human genome. The disadvantage of this system is that oligonucleotides have a very short half-life and do not enter cells readily. However, these are technical problems which can and are being solved. Viral infections and cancer are two acquired diseases where antisense therapy holds the greatest promise. For example, RNA antisense oligonucleotides against the envelope gene of HIV were encapsulated in liposomes (to facilitate cell uptake and to protect the oligo from nucleases) and targeted for HIV host cells. HIV replication was inhibited by 91% using env antisense and only marginally using sense env. To get around the problem of the short half-life of oligos, another strategy would be to construct a plasmid which contains DNA in the antisense orientation for the full length cDNA of a viral protein. When cells are transfected with a plasmid encoding antisense F (RSV fusion protein), RSV replication in the cell is reduced 100-fold. In summary, by knowing specific molecular mechanisms of an acquired lung disease, very specific gene therapy can be designed whereby the product of the gene acts as a drug to ameliorate the disease. Because permanent transgene expression is not needed, safer, non-viral vectors can be used for transient transgene expression. Back to Top GENE THERAPY FOR ACUTE LUNG INJURY BASIS FOR GENE THERAPY Our understanding for the pathogenesis of acute lung injury has improved but mortality has remained the same. Delivery of a gene(s) which encodes for a protein(s) that decrease the susceptibility of lung cells to injury or hasten their recovery. POTENTIAL GENES Gene Mechanism of Action *Alpha1 Antitrypsin Antiprotease Mn superoxide dismutase antioxidant (mitochondrial) Cu-Zn superoxide dismutase antioxidant (cytoplasmic) Cyclooxygenase (PGH Synthase) catalyzes production of prostanoids *Cytokines TNF receptor (p55) IL-10 Inhibits effect of cytokine or increases production of "protective cytokine" lKB Inhibits activation of NFKB Human Tumor Necrosis Factor Receptor (p55) and Interleukin 10 Gene Transfer in the Mouse Reduces Mortality to Lethal Endotoxemia and Also Attenuates Local Inflammatory Responses Materials and Methods Animal Model: Mice Plasmids: pCMVp55 Plasmid containing the open reading frame for human p55 (soluble TNF receptor) pcD-SR-IL-10 Plasmid containing the open reading frame for human IL-10 Gene Transfer: 200(g DNA complexes to 100(M DDAB:DOPE delivered intraperitoneally Endotoxin Challenge: 48 hours after gene transfer, the mice were given 250 ng E. coli LPS and 18 mg D-galactosamine Lethality in this model is dependent on TNF response RESULTS Message and gene product was detected in the lung, liver, spleen, and kidney. The highest expression was seen in the spleen; the lowest was seen in the lung. IL-10 gene transfer decreased serum TNF levels Experiment No pCMV/p55 pcD-SR-a/hIL10 Irrelevant DNA/Liposomes SL Survived/total Survived/total Survived/total 1 4/6 6/6 1/6 2 3/6 4/6 0/6 3 3/6 6/6 1/6 Totals 10/18 16/18 2/18 TNF-a pg/ml 2080+/- 810 190+/- 60 2690+/-660 PGH SYNTHASE GENE THERAPY PGH Synthase catalyzes the conversion of arachidonic acid to protanoids. It is the rate limiting step in this conversion. Prostacyclin and PGE2 are the principle products generated by endothelial cells. Large vessel pulmonary endothelial cells produced predominantly prostacyclin. Microvascular pulmonary endothelial cells produced predominantly PGE2. Prostacyclin is a vasodilator: PGE2 has numerous anti-inflammatory actions Theory: A small increase in the expression of PGH synthase in pulmonary endothelia cells could result in a marked increase in the local synthesis of prosacyclin and PGE2 that may result in protection against the physiologic effects of endotoxin. Methods Animal model: New Zealand White Rabbits Plasmids: pCMV4PGH pCMV4 without gene insert Gene Expression: 24 hours after gene transfer, rabbits were killed and one gram of lung was obtained for 24 hour organ culture. Prostanoid production was measured in the media. Endotoxin Challenge: 24 hours after transfection, a in situ-perfused lung preparation was established. After 30 minutes, a single bolus of LPS (4(g/kg) was given and pulmonary artery pressure was monitored. Results PGH Synthase gene transfer resulted in an increased production of prostacyclin and PGE2 in lung organ cultures 24 hours after transfection. PGH Synthase gene transfer blocked the progressive increase in mean pulmonary artery pressure following LPS exposure. Thromboxane B2 was decreased in the lung perfusate after LPS administration in the animals that received PGH Synthase. a 1AT Gene Therapy for Acute Lung Injury Pig in situ Lung Model of Endotoxin Induced Acute Lung Injury 48 hours before endotoxin, pCMVa1AT or pCMVCAT is administered through a central vein (jugular) 1 mg/kg DNA is given DNA complexed 1:3 (w/w) with DOTMA/DOPE Sterile H2O is added to DNA to equal volume of lipid At the time of endotoxin challenge, the in situ preparation is established and endotoxin infused. Measurements: Pulmonary artery pressure Left atrial pressure Pulmonary blood flow WBC Temperature and blood gasses are kept constant Calculations: Distribution of DNA RNA a1AT serum, BAL, and organ culture levels IL-8 Elastin peptide fragments Immunohistochemistry and In situ hybridization a1AT Gene Therapy for Acute Lung Injury Pig in situ Lung Model of Endotoxin Induced Acute Lung Injury Serum Levels of a1AT 48 Hours After Gene Transfer Average 228.9 ng/ml (range 57.6-787.4 ng/ml) Although detectable, these levels are significantly below what is normally considered therapeutic Conclusions Gene Therapy may play a role in future medical interventions for Acute Lung Injury. Several gene have the potential to be beneficial but at this point all the genes are targeting the inflammatory component of lung injury. Gene that encode for cytokines - p55 and IL-10 modulate the dramatic cytokine cascade seen in ALI. PGH Synthase increases production of beneficial prostanoids that may limit the physiological response to endotoxin as well as the inflammatory response. Alpha1 antitrypsin attempts to decrease the protease burden, potentially in the microenviroment of the neutrophil-endothelial interface or even possibly by increasing the intracellular levels of the antiprotease. Back to Top GENE THERAPY FOR INHERITED LUNG DISEASES The two most common hereditary lung disorders in individuals of European descent are cystic fibrosis (CF) and alpha, antitrypsin (a1AT) deficiency. Both of the human genes have been cloned and gene therapy has a potential role in the treatment of both of these diseases. Ex vivo delivery strategies targeting the lung is not an efficient delivery system for several reasons. First, it is very difficult to grow "normal" respiratory epithelial cells in culture to transduce. Second, most ex vivo strategies use retroviruses to transduce the cells. Retrovirus requires active dividing cells to infect and the majority of respiratory cells are terminally differentiated. Third, the total surface area of the lung is 140 m2 (airways are 1-2 m2) and there are 23 generations of dichotomous branching tubes. a1AT is normally made in the liver. For a1AT deficiency, some investigators are targeting the liver with retroviruses rather then targeting the lung. In this case, ex vivo gene therapy is an option but the targeted organ is the liver, not the lung. Most gene therapy studies involving the lung have focused on in vivo gene delivery systems, mainly adenoviral vectors and plasmid-liposome complexes. Both can be delivered either intravenously or by aerosol with expression in the lung. The advantages of the adenovirus is its efficiency at gene delivery. It disadvantage is its toxicity - humoral, cellular, and neurogenic. The advantages to plasmid-liposome complexes are the ease of production and the low toxicity profile. The disadvantage is its low efficiency. More recent work has focused on the adeno-associated virus and on plasmid-virus hybrids where investigators are trying to exploit the strengths of both systems. Back to Top CYSTIC FIBROSIS CF is an autosome recessive disorder caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. It is the most common, lethal hereditary disorder in the USA. It occurs predominantly in Caucasian and Hispanic populations. 1 in 20 are heterozygous carriers; the incidence of clinical disease is 1 in 2500 live births. Heterozygous carriers have no clinical manifestations of CF. The most common mutation is delta-F508, a deletion of three nucleotides resulting in the loss of a phenylalanine a residue 508. This deletion is responsible for 60-70% of cases of CF. The CFTR protein is a Cl- channel that modulates the secretions of Cl- in response to elevations of intracellular cyclic AMP. Mutations of the CTFR gene prevent epithelial cells from performing this function. This results in a higher than normal transepithelial potential difference of the tracheobronchial tree. CFTR has been localized to the apical membranes of pancreatic ducts, intestinal epithelia, sweat ducts, and airway epithelia including columnar, ciliated, and mucous-producing cells. There is also indirect data suggesting the CFTR may be localized to organelles, including the Golgi apparatus. The clinical manifestations are primarily in the lung, liver, GI tract, and pancreas. Over 90( of deaths in CF are due to respiratory failure and most of the work in gene therapy has focused on correcting the genetic defect in the lungs. Hepatobiliary disease is the second leading cause of death. The general concept on how CFTR causes lung disease is that the defect in CFTR prevents adequate hydration of the epithelial lining fluid. This causes mucous obstruction with subsequent infection and inflammation. This is probably an oversimplified concept. Currently, there is no cure for CF. Therapies for CF are time-consuming, intensive, and expensive. The average cost of medical therapy is 27,500/year/patient. About 40% of CF individuals are hospitalized 1-2 times per year. The average life-span is approximately 28 years. Rationale for Gene Therapy The association between the defect in the CFTR gene and lung disease is clear. How this defect causes lung disease is not clear. There are two general hypotheses: External electrolyte milieu. The respiratory manifestations result from changes in the electrolyte milieu on the surface of the airway epithelium. This suggests that most or all of the airway epithelium is involved with CFTR expression. Internal milieu. The respiratory manifestations result from processes internal to the respiratory epithelial cells. This suggests that a subset of airway epithelial cells (e.. mucous-secreting cells) play a dominant role. This distinction may be important because mucous producing cells are the dominant cell responsible for the lung disease and gene delivery to these cells is more efficient when administered intravenously. CFTR gene expression is very low, averaging 1-2 mRNA transcripts per cell and only about 10% of cells need to be corrected in order to correct the abnormal potential difference. [CLICK HERE FOR MORE] Back to Top Alpha 1 Antitrypsin Deficiency a1AT deficiency is a genetic disease seen predominantly in individuals of European descent and is second only to CF as the most common, lethal, hereditary disorder in the U.S. It is autosomal codominant in which hepatocytes fail to secrete adequate amounts of the protein. The Z mutation results in intraepatic accumulation of the protein, low serum levels, and is responsible for the vast majority of a1AT deficiency. Deficiency is defined as 35 % of normal serum concentrations. However, a 'critical threshold' of 10-15% of normal seems necessary to place an individual at risk for emphysema. It is synthesized predominantly in the liver but some synthesis occurs in the monocyte-macrophage system. It is the major antiprotease found in humans and forms the bulk of the antiprotease protection in the lower respiratory track. The major organs effected by a1AT deficiency are the lungs- due to the loss of antiprotease protection- and the liver- due to direct toxic effect of a1AT accumulation. One normal allele obviates any risk from the second allele for emphysema. The most prominent clinical feature of hereditary emphysema, aside from the young age at presentation, is the marked destruction of the lower zones of the lungs. This pattern of destruction is observed in 95% of patients with hereditary emphysema. This is in contrast to the upper zone or more uniformly distributed disease seen in smokers with acquired emphysema. Pathologically, a1AT deficiency is associated with the panacinar form of emphysema. Again this is in contrast with acquired emphysema in which centrilobular emphysema generally dominates. The discovery of a1AT uncovered a whole family of enzymes called serine proteases or Serpins. Serpins are single-chained proteins with there reactive center located on an exposed loop and their inhibitory specificity primarily defined by a single amino acid at the reactive center of this loop. The reactive site for a1AT is Methionine at position 358. a1AT reacts with neutrophil elastase to form a stable, inactive enzyme-inhibitor complex. During the inhibition process, irreversible changes in the 3 dimension structure of a1AT occurs resulting in suicidal binding of a1AT to elastase. The Achilles heel of a1AT is its susceptibility to oxidation. Oxidation at its reactive site decreases its association with elastase by 2000 fold. Oxidation of a1AT may be a contributing factor in the pathogenesis of acquired emphysema. The discovery of a1AT deficiency revolutionized our understanding of emphysema. Before 1962 most physicians believed that the destruction of alveolar surface found in emphysema was a reflection, rather than the cause of airway obstruction. The discovery of a1AT and the observation that elastase can produce emphysema when instilled into the lungs spawned the Protease Theory of Emphysema. This theory proposes that derangement in the normal homeostasis between proteases and antiproteases results in emphysema. Gene Therapy for a1AT deficiency There are two main strategies for a1AT gene therapy. One strategy is to target the liver where the protein is normally synthesized. This can be done by isolating hepatocytes followed by ex vivo transduction with a retrovirus containing the a1AT gene and re-infusing (usually through the splanchic vein) back to the liver. Another Approach has been to perform a partial hepatectomy followed by in vivo transduction with retroviruses. The third approach is direct in vivo gene delivery with adenoviral vectors or plasmid-liposome complexes. The second strategy is to target the lung directly where a1AT forms the bulk of the antiprotease protection in the lower airways. Delivery to the lungs can be either intravenously or be aerosol. Both adenoviral vectors and the plasmid-liposome complexes have been used. Back to Top SURFACTANT PROTEIN DEFICIENCY Surfactant Protein B Deficiency Human surfactant protein B (SP-B) is a phospholipid-associated polypeptide expressed by respiratory epithelial cells. It is essential for lung function, enhancing the spreading and stability of surfactant phospholipids that reduce surface tension at the alveolar air-liquid interface. Surfactant protein B deficiency is an autosomal recessive disease of full-term newborn infants which leads to lethal respiratory failure within the first year of life. Rationale for gene therapy for surfactant protein B deficiency. This disorder therefore represents a logical candidate for gene therapy, in which it is proposed that human SP-B cDNA be transferred to the epithelial cells of the lower respiratory tract. To this end, two groups have demonstrated pulmonary expression of SP-B following adenoviral-mediated gene transfer to the respiratory epithelium of rodents.[22,23] Other investigators have hypothesized that because SP-B deficiency manifests in the perinatal period, prenatal or fetal gene therapy might be required to minimize morbidity. In this approach, it will be necessary to optimize the timing of gene transfer to maximize gene expression in the airways while minimizing the potential inflammatory response that may be mounted by the immune system of the developing fetus. Conclusion Future Challenges Although tremendous progress has been made in the ability to successfully transfer genes to cells, the field of gene therapy is still in its infancy. While many therapeutic genes have been identified for the treatment of both inherited and acquired diseases of the lung, there remain several unresolved problems limiting the practical translation of these gene therapy strategies at the present time. A number of researchers are attempting to increase the transduction efficiency of viral and nonviral vectors in order to permit gene transfer to a greater percentage of cells. To this end, strategies are also being explored which would enable viral vectors to replicate specifically within the target cells -- to permit gene transfer within a solid tumor, for example. Other investigators are seeking to develop vector targeting strategies important in transferring genes to disseminated cancer cells as well as to specific cells in compartmentalized models of disease. Methods to regulate the expression of therapeutic genes are being explored and it is expected that much attention will be focused on the identification of promoters which can selectively activate genes only within the target disease cells. Other key areas of future research include modifying viral vectors to reduce the toxicity and immunogenicity associated with their use. Further development of vector delivery systems will also involve, for example, modifications to adenoviral vectors to permit long-term gene expression, which will be important for the application of gene therapy in the management of certain more chronic diseases. The lung will continue to serve as a model system in the field of gene therapy in which the requirements for advances in vector development are identified and novel vectors are evaluated. As vector technology evolves, it should become possible to realize the full potential of gene therapy as a valid therapeutic option for pulmonary diseases.

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