Clinical potential of gene therapy: towards meeting the demand.

Since the discovery that new genetic material could be transferred into human cells resulting in induced expression of genes and proteins, clinicians and scientists have been working to harness the technology for clinical outcomes. This article provides a summary of the current status of developments within the broad discipline of clinical gene therapy. In pursuing the treatment of diverse clinical conditions, a wide variety of therapeutics, each tailor-made, may be required. Gene therapy offers the possibility of accurately and specifically targeting particular genetic abnormalities through gene correction, addition or replacement. It represents a compelling idea that adds a new dimension to our portfolio of credible therapeutic choices.

Ribozymes in gene therapy of HIV-1.

Human immunodeficiency virus type 1 (HIV-1) is the primary etiologic agent for Aquired Immune Deficiency Syndrome (AIDS). HIV-1 is a lentivirus, a separate genus of the Retroviridae, which are complex RNA viruses that integrate into the genome of host cells and replicate intracellularly. Ribozymes are catalytic RNA molecules with enzyme-like cleavage properties, that can be designed to target specific RNA sequences within the HIV-1 genome. In addition to the genomic RNA, several RNA intermediates, including splice variants, can be targeted by a single ribozyme. We and others have demonstrated the ability of ribozymes to suppress HIV-1 replication in a variety of cultured cells. Ribozyme gene therapy for HIV-1 infection is a therapeutic approach that offers several potential advantages over conventional therapies in that it can potentially impact on both viral load and restoration of the immune system. Ribozyme gene therapy may be used as an adjunct to chemotherapeutic drugs, effecting viral suppression, and facilitating immune restoration without problems of patient compliance. Currently, an anti-HIV-1 ribozyme is being tested in two separate Phase I Clinical Trials.

Cryoprecipitated antihemophilic factor production from blood collected in quad packs or from blood with delayed processing. The importance of plasma thawing method.

Two alternate approaches to increasing the supply of plasma suitable for the production of cryoprecipitated AHF (cryo) were evaluated. In the first, cryo was prepared from blood collected in quadruple packs from which red cells and platelets also were made. This procedure resulted in a mean reduction of starting plasma volume of about 25 percent with a concomitant decrease in factor VIII coagulant activity (FVIII:C) of cryo when compared to approximately 2000 cryos made in triple packs assayed in our laboratory during the past year. However, American Red Cross (ARC) regions using a higher yield waterbath method of thawing plasma successfully produced quadruple pack cryos with a mean potency of 109 international units per container and about 90 percent of them met federal potency requirements. This was not the case for ARC regions using the lower yield method of thawing plasma in a refrigerator. The second approach involved cryo production from plasma frozen following 15 hours of cold storage of either the separated plasma or whole blood. Cold storage of plasma resulted in small but insignificant decreases in FVIII:C potency and yield in the cryo. However, there was a 20 percent decrease in FVIII:C yield in cryo produced from frozen plasma following 15 hours of cold storage of whole blood. Again, the decrease in FVIII:C yield resulting from interim blood storage was compensated for by the use of the higher yield waterbath method.

The acquisition of granylocytes by leukapheresis: a comparison of continuous flow centrifugation and filtration leukapheresis in normal and corticosteroid-stimulated donors.

Leukapheresis by continuous flow centrifugation (CFC) or filtration (FL) were compared in untreated and corticosteroid-treated donors. The administration of prednisone 60 mg orally 10 to 12 hours before leukapheresis increased significantly both the donor's prepheresis WBC counts and the total granulocyte yields by CFC or FL. Dexamethasone 6 mg iv at the start of FL did not increase granulocyte yields significantly. In untreated donors FL yielded 0.25 x 10(10) granulocytes per liter donor blood processed as compared with 0.05 x 10(10) per liter in CFC donors. Corticosteroid premedication has a greater relative effect in increasing yields by CFC than by FL. Donor reaction rates were between 4 and 6 per cent for both procedures.

Plateletpheresis using the haemonetics model 30 cell separator.

Plateletpheresis using the Haemonetics Model 30 cell separator results in a mean collection of 5.5 X 10(11) platelets. This product contains substantial numbers of erythrocytes and mononuclear cells which can be effectively removed by centrifugation but with a 23% loss of platelets as well. The mean decrement in donor's platelet count during the procedure, which is 78,000/mm3, is not commensurate with the number of platelets collected suggesting donor platelet mobilization. Platelets collected by this technic, when transfused into unselected recipients, give platelet increments comparable to platelets produced in the standard batch manner.

Inhibition of granulocyte erythrophagocytosis by HLA antisera.

The ability of HLA antisera to inhibit granulocyte erythrophagocytosis (EP) of opsonized red blood cells (RBC) was evaluated. Human granulocytes (PMN) were separated from heparinized whole blood by the Ficoll-Isopaque technique and suspended in McCoy's medium. EP occurred when the PMN were incubated with opsonized RBC. For six HLA antibody specificities evaluated, prior incubation of PMN with HLA antisera resulted in significant inhibition of EP without cytolysis when the PMN donor was positive for the specific HLA antigen, but not when the donor was negative for the antigen. The inhibition was time- and dose-dependent. Prior absorption of HLA antisera with HLA-specific platelets reduced or abolished the inhibition. An example of anti-NB1 also inhibited EP in 4 individuals. These data suggest that HLA antibody may adversely affect granulocyte phagocytic function. Inhibition of EP might be useful in evaluating compatibility prior to granulocyte transfusion.

Plateletpheresis residues: a source of large quantities of human blood lymphocytes.

The residue from single-donor plateletpheresis contains a large number of human mononuclear cells. We have been able to harvest more than 1 X 10(9) viable lymphocytes for laboratory study from the leukocyte-rich sediment that previously had been discarded. Prior to removal by adherence 5 X 10(8) monocytes were also available for future purification, if desired. The physical properties and response to phytohemagglutinin were very similar when lymphocytes isolated from plateletpheresis residues were compared with those obtained directly from veneous blood.

Application of biosafety principles in blood establishments.

In light of increasing public and employee concern over potential infectious hazards associated with blood and other body fluids, several government agencies (the Food and Drug Administration, the Centers for Disease Control, the Occupational Safety and Health Administration, the Environmental Protection Agency, the Health Care Financing Administration and the National Heart, Lung and Blood Institute) cosponsored a Biosafety Workshop in April 1988. The objective of the workshop was to identify appropriate biosafety practices and standard control procedures to protect workers involved in the collection, storage, and transportation of human blood donations with the least possible disruption of the nation's blood supply. Speakers focused on human immunodeficiency virus (HIV) and hepatitis B virus (HBV); however, the safety principles discussed were considered equally applicable to other known (e.g., non-A, non-B hepatitis and human T-lymphotropic virus type I (HTLV-1) blood-transmitted infections. The resulting consensus included the need for blood establishments to develop and apply thoughtful biosafety programs to address staff training, accident prevention, HBV vaccination, handling spills, managing contaminated waste and transporting blood specimens. There was lack of agreement, however, on the usefulness of gloves during the phlebotomy of healthy blood donors.

Granulocyte kinetics in donors undergoing filtration leukapheresis.

Normal blood donors undergoing filtration leukapheresis (FL) have a profound transient neutropenia early in the procedure which is followed by a "rebound" neutrophilia. This phenomenon occurs in unstimulated donors as well as in donors pretreated with either prednisone or dexamethasone. The mechanism for development of the neutropenia was investigated in volunteers throug a nylon filter at 37 C, a significant but transient neutropenia was observed. Plasma rendered cell and stroma-free achieved the same result indicating that plasma alone, when exposed to nylon fibers, is capable of producing neutropenia.

Abdominal pain in donors during filtration leukapheresis.

An unusual donor reaction characterized primarily by lower abdominal and/or perineal pain occurs in 1.2 per cent of donors during filtration leukapheresis (FL). The reaction occurs almost exclusively in female donors. Corticosteroid pretreatment appears to be highly effective in preventing the reaction. The pathogenesis of this donor problem is speculative.

The use of ribozyme gene therapy for the inhibition of HIV replication and its pathogenic sequelae.

Human immunodeficiency virus (HIV) is a lentivirus, a separate genus of the Retroviridae which are RNA viruses that integrate as DNA copies into the genomes of host cells and replicate intracellularly through various RNA intermediates. Several of these RNA molecules can be targeted by ribozymes and a number of investigators, including our group, have demonstrated the ability of ribozymes to suppress HIV replication in cultured cells. It is argued that the use of this ribozyme gene therapy approach for the treatment of HIV infection may act as an adjunct to chemotherapeutic drugs and may affect not just viral suppression, but also immune restoration. This approach can be tested in Clinical Trials, several of which are currently under way.

Continuous release of secretory granule proteoglycans from a cell strain derived from the bone marrow of a patient with diffuse cutaneous mastocytosis.

A human cell strain (designated HBM-M) that was derived from the bone marrow of a child with diffuse cutaneous mastocytosis was previously found to possess features that suggested it belonged in the mast cell/monocyte lineage. HBM-M cells synthesized approximately 150-Kd Pronase-resistant proteoglycans that were recognized by an antihuman secretory granule proteoglycan peptide core antibody. These cells also contained in relatively high abundance the same sized mRNA transcript that encodes the peptide core of proteoglycans that are normally localized to secretory granules of hematopoietic cells. However, unlike most other hematopoietic cells, HBM-M cells continuously released their newly synthesized 35S-labeled proteoglycans rather than retaining them in an intracellular storage compartment. Chondroitinase ABC, nitrous acid, and heparinase degraded approximately 76%, 17%, and 7%, respectively, of the HBM-M cell-derived 35S-labeled proteoglycans. As assessed by high performance liquid chromatography, 91% of the unsaturated 35S-labeled disaccharides generated by treatment with chondroitinase ABC were delta Di-4S. The remaining chondroitin sulfate 35S-labeled disaccharides appeared to be primarily a complex mixture of disulfated disaccharides. The 35S-labeled glycosaminoglycans that were not degraded by chondroitinase ABC migrated in two-dimensional cellulose acetate electrophoresis as if they were heparan sulfate or under-sulfated heparin. Thus, although the HBM-M cell-derived proteoglycans had some of the features of proteoglycans produced by normal human mast cells, the heparin-like and chondroitin sulfate glycosaminoglycans bound to the HBM-M cell proteoglycans were considerably less sulfated. Because the only human cell types that have so far been shown to synthesize proteoglycans that have heparin-like glycosaminoglycans bound to a protease-resistant peptide core are mast cells and basophilic leukocytes from patients with myelogenous leukemia, it is possible that the HBM-M cell is a mast cell progenitor cell.

Complications related to equipment and technique.

Automated cytapheresis, when performed by experienced operators using modern equipment, is remarkably free of adverse effects. A declining pattern of equipment failure and related problems has been recorded in the 220,000 procedures performed by the American Red Cross in the past 5 years. Other cytapheresis centers report similar findings. Current CBCS are safe and reliable, and defects in plastic disposables, while irritating, result in little danger to the donor. Operator vigilance and careful attention to the details of the procedure can eliminate the majority of serious potential complications that have been encountered to date.

Small bowel mucosa from celiac patients generates 15-hydroxyeicosatetraenoic acid (15-HETE) after in vitro challenge with gluten.

Celiac disease (gluten-sensitive enteropathy [GSE]) is a disorder characterized by small intestinal mucosal injury caused by dietary exposure to wheat gluten and similar proteins. There is evidence that the mucosal injury is immunologically mediated and there is an inflammatory infiltrate present in the mucosa. It is postulated that release of lipid-derived inflammatory mediators may be involved in the pathogenesis of the mucosal injury. Jejunal mucosal biopsy samples from patients with GSE and from a group of patients who were subsequently shown to have normal jejunal mucosa were incubated with tritiated arachidonate and a peptic/tryptic digest of either gluten or casein. Generation of lipid-derived inflammatory mediators was measured by beta-scintillation counting after separation of metabolites by reverse-phase high performance liquid chromatography with two different buffer systems. The predominant arachidonic acid metabolite generated was 15-hydroxyeicosatetraenoic acid (15-HETE). Mucosa from newly diagnosed GSE patients on a normal diet generated more 15-HETE than either control patients or GSE patients maintained on a gluten-free diet. In addition, gluten acted as a specific stimulus to 15-HETE production by mucosa from the GSE patients on a normal diet. 15-HETE has a number of biologic effects that could contribute to the mucosal changes seen in GSE, and the specific release of 15-HETE by gluten suggests involvement in the pathogenesis of the disorder.

Occurrence of platelet-activating factor (PAF) and an endogenous inhibitor of platelet aggregation in diffuse cutaneous mastocytosis.

We have identified PAF in the blister fluid from a patient with bullous mastocytosis, a rare form of mast-cell disease. We have found a novel endogenous inhibitor of platelet aggregation which obscured the presence of the PAF in unprocessed blister fluid and in ethanol or lipid extracts. The PAF was characterized by the demonstration of chromatographic, mass spectral and biological properties identical to those of authentic PAF. Thus this is the first demonstration of PAF in biological fluid from a patient with mastocytosis. High levels of immunoreactive prostaglandin D2 (PGD2) and histamine were also present in the blister fluid. The interaction between PAF and the inhibitor of platelet aggregation in patients with systemic mastocytosis may provide an explanation for some of the manifestations of the disease, in particular the episodic hypotension, cutaneous flushing and pallor.

Production and characterization of antibodies to platelet-activating factor.

Antibodies directed against platelet-activating factor (PAF) have been raised in rabbits immunized with a novel platelet-activating factor analogue-conjugate. An analogue of PAF with a carbon double bond at the terminal end of the alkyl chain was subjected to ozonolysis and converted to the aldehyde. The aldehyde was coupled to thyroglobulin by reductive alkylation and rabbits were immunized via either intramuscular or intradermal routes in complete Freund's adjuvant. The antibodies are specific for PAF with a preference for the optically active (R)-enantiomer. There appears to be a requirement for antibody binding of an alkyl chain of up to 18 Carbon atoms at C1, and an acetyl group in the C2 position. The ability of a number of structural analogues to inhibit binding of tracer to the antibody is related to the biological activity of the analogue, and therefore may reflect the structural domains that are critical for PAF to interact with its receptor.