STRATEGIES IN GENE THERAPY
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.
GENE THERAPY FOR CANCERMany 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. 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 GeneMetastatic 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 CellsA 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.
|
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:
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:
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.
Average 228.9 ng/ml (range 57.6-787.4 ng/ml)
Although detectable, these levels are significantly below what is normally
considered therapeutic
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.
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.
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.
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:
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.
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.
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.
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.
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.