Clinical Gene Transfer Studies Research Articles

An International Epidemiology Study to Determine the Prevalence of Preexisting Cell-Mediated Immunity to Adeno-Associated Virus (AAV) in Adults with Severe Hemophilia

Introduction: Adeno-associated virus (AAV) is a versatile gene therapy vector utilized widely in clinical gene transfer studies. AAV serotype 8 (AAV8) has been used successfully as a vector in liver-directed gene transfer in hemophilia clinical trials, and a recombinant AAV8 vector is used currently in Shire's BAX 888 clinical study (NCT03370172) for the treatment of patients with severe hemophilia A. However, 1 recognized limitation of intravenous administration of recombinant AAV is the barrier posed by preexisting immunity against AAV and the cross-reactivity of anti-AAV antibodies between various serotypes that results from natural exposure to the wild-type viruses. Neutralizing antibodies (NAbs) resulting from humoral immune responses can compromise vector transduction, and reactivation of memory T cells from cell-mediated immune responses can abrogate transgene expression and eliminate transduced cells. Understanding the prevalence of cell-mediated immune responses in the hemophilia population and the association between cell-mediated and humoral immunity is important in developing effective gene therapies.Objectives: To determine the prevalence of preexisting immunity towards AAV8 and AAV2 in the adult population of patients with hemophilia A and B, and to characterize the association between cell-mediated immunity and humoral immunity.Methods: In this ongoing study, peripheral blood samples were collected at outpatient visits from adult males (age 18-75 years) with severe hemophilia A (plasma factor VIII [FVIII] activity <1%) or hemophilia B (plasma factor IX [FIX] activity ≤2%) for determination of immunity towards AAV8 and AAV2. Patients could elect to participate in a single baseline visit or in the longitudinal study arm for up to 3 annual visits. Patients with inhibitors or a history of inhibitors to FVIII or FIX, an underlying lymphocyte disorder, or currently receiving cytotoxic chemotherapy, monoclonal antibodies, immunoglobulins, immunosuppression, or systemic antiviral therapy were excluded. The cell-mediated immune response against AAV8 peptide antigens was quantified using an interferon-γ enzyme-linked ImmunoSpot (ELISPOT) assay to determine the number of T cells responsive to 3 different pools of AAV8 capsid-specific protein fragments. Each AAV8 antigen pool consisted of 50-60 nonoverlapping peptides composed of 15 amino acid residues. Results were considered positive if the signal was >3 times above the background and >60 cells/million. Serum was collected for assessment of humoral immunity (AAV8 NAbs or AAV2 NAbs) using a cell-based transduction inhibition assay (titer cutoff: ≥1:5).Results: A total of 41 patients have enrolled in the study to date (mean ± SD age: 33.8 ± 11.1 years) and ELISPOT data are available for 37 patients. Of those with available data, 9/37 patients (24.3%) had a positive ELISPOT to ≥1 of the 3 AAV8 peptide pools tested. One patient tested positive for peptide pool 1, 3 for peptide pool 2, and 7 for peptide pool 3 (2 patients [5.6%] tested positive for both pools 2 and 3). Of the 9 patients with positive AAV8-specific ELISPOT results, 6 (66.7%) also had a positive result for AAV8 NAbs, while of 27 patients with negative ELISPOT results, 11 (40.7%) had a positive result for AAV8 NAbs.Conclusions: The results from this ongoing international epidemiology study demonstrate that the prevalence of preexisting cell-mediated immunity to AAV8 resulting from natural exposure to wild-type AAV is approximately 1 in 4 adult males with severe hemophilia A or B. Furthermore, there appears to be a correlation between cell-mediated and humoral immune responses, as two thirds of patients with positive AAV8-specific ELISPOT results also tested positive for AAV8 NAbs. Understanding the relationship between cell-mediated and humoral immunity and how it may affect an individual's response to AAV-based gene therapy will be key to successfully developing this novel therapy. DisclosuresRajavel:Shire: Employment, Equity Ownership. Chapin:Shire: Employment, Equity Ownership. Tang:Shire: Employment.

Directed Engineering of a High-expression Chimeric Transgene as a Strategy for Gene Therapy of Hemophilia A

Human coagulation factor VIII (fVIII) is inefficiently biosynthesized in vitro and has proven difficult to express at therapeutic levels using available clinical gene-transfer technologies. Recently, we showed that a porcine and certain hybrid human/porcine fVIII transgenes demonstrate up to 100-fold greater expression than human fVIII. In this study, we extend these results to describe the use of a humanized, high-expression, hybrid human/porcine fVIII transgene that is 89% identical to human fVIII and was delivered by lentiviral vectors (LVs) to hematopoietic stem cells for gene therapy of hemophilia A. Recombinant human immunodeficiency virus-based vectors encoding the fVIII chimera efficiently transduced human embryonic kidney (HEK)-293T cells. Cells transduced with hybrid human/porcine fVIII encoding vectors expressed fVIII at levels 6- to 100-fold greater than cells transduced with vectors encoding human fVIII. Transplantation of transduced hematopoietic stem and progenitor cells into hemophilia A mice resulted in long-term fVIII expression at therapeutic levels despite <5% genetically modified blood mononuclear cells. Furthermore, the simian immunodeficiency virus (SIV) -derived vector effectively transduced the human hematopoietic cell lines K562, EU1, U.937, and Jurkat as well as the nonhematopoietic cell lines, HEK-293T and HeLa. All cell lines expressed hybrid human/porcine fVIII, albeit at varying levels with the K562 cells expressing the highest level of the hematopoietic cell lines. From these studies, we conclude that humanized high-expression hybrid fVIII transgenes can be utilized in gene therapy applications for hemophilia A to significantly increase fVIII expression levels compared to what has been previously achieved.

Prolonged Susceptibility to Antibody-mediated Neutralization for Adeno-associated Vectors Targeted to the Liver

Adeno-associated virus (AAV) vectors demonstrate highly efficient gene transfer to hepatocytes in vivo. One of the remaining obstacles to the treatment of hemophilia B patients with AAV vectors is the sensitivity of these vectors to antibody-mediated neutralization following systemic delivery. Testing and implementation of strategies to circumvent pre-existing antibodies requires knowledge of the clearance kinetics of AAV from circulation. In this study, AAV clearance kinetics were established for serotypes 2 and 8 in cell culture and in mice. Administration of pooled neutralizing serum subsequent to administration of the vector was used to define the time period in which the vector is susceptible to antibody-mediated neutralization. These experiments defined the in vivo clearance rates for both AAV2 and AAV8 vectors to be between 2 and 4 hours. In mice, portal vein and tail vein administration of each vector was tested with similar results. Cell culture studies in W162 cells established that cellular attachment and internalization both contribute to the clearance kinetics of AAV vectors. These studies characterize the in vivo clearance rates of AAV vectors for the first time and guide the development of future strategies for the avoidance of antibody-mediated AAV vector neutralization.

Immune Responses to Adenovirus and Adeno-Associated Vectors Used for Gene Therapy of Brain Diseases: The Role of Immunological Synapses in Understanding the Cell Biology of Neuroimmune Interactions

Researchers have conducted numerous pre-clinical and clinical gene transfer studies using recombinant viral vectors derived from a wide range of pathogenic viruses such as adenovirus, adeno-associated virus, and lentivirus. As viral vectors are derived from pathogenic viruses, they have an inherent ability to induce a vector specific immune response when used in vivo. The role of the immune response against the viral vector has been implicated in the inconsistent and unpredictable translation of pre-clinical success into therapeutic efficacy in human clinical trials using gene therapy to treat neurological disorders. Herein we thoroughly examine the effects of the innate and adaptive immune responses on therapeutic gene expression mediated by adenoviral, AAV, and lentiviral vectors systems in both pre-clinical and clinical experiments. Furthermore, the immune responses against gene therapy vectors and the resulting loss of therapeutic gene expression are examined in the context of the architecture and neuroanatomy of the brain immune system. The chapter closes with a discussion of the relationship between the elimination of transgene expression and the in vivo immunological synapses between immune cells and target virally infected brain cells. Importantly, although systemic immune responses against viral vectors injected systemically has thought to be deleterious in a number of trials, results from brain gene therapy clinical trials do not support this general conclusion suggesting brain gene therapy may be safer from an immunological standpoint.

Utility of PEGylated recombinant adeno-associated viruses for gene transfer

Adeno-associated virus (AAV), capable of producing significant, long-term transgene expression, is one of the least toxic vectors employed in pre-clinical and clinical studies of gene transfer. One limitation is generation of neutralizing antibodies against viral capsids, blocking gene expression after readministration. AAV2 capsids were modified with poly(ethylene) glycols (PEGs) activated by cyanuric chloride (CCPEG), succinimidyl succinate (SSPEG) and tresyl chloride (TMPEG). SSPEG and TMPEG conjugation did not compromise gene transfer to the liver and muscle and improved gene expression in the lung 5 fold. Transduction efficiency of CCPEG-AAV was impeded in all tissues by aggregation. TMPEG afforded the best protection from neutralization in vitro and in vivo. Gene expression in mice immunized against unmodified AAV was reduced by a factor of 10 from that of naïve animals after intramuscular rechallenge with PEGylated AAV but was not significantly different from naïve mice after intravenous readministration ( p = 0.08). Gene expression was markedly reduced in muscle after two doses of PEGylated AAV. In contrast, mice given two intravenous doses of TMPEG-AAV had significantly higher transgene levels than naïve animals 14 days after rechallenge ( p = 0.001). This technology could promote successful readministration of vector in the clinic and marked expression in patients with anti-AAV antibodies.

397. AAV-Mediated Gene Transfer to Endothelial Cells

Adeno-associated virus (AAV) vectors have been evaluated in clinical studies of gene transfer for hemophilia by two different strategies, one using liver as the target tissue and the other using skeletal muscle. Both routes have resulted in long-term cures in hemophilic dogs. In humans, delivery to skeletal muscle resulted in long-term expression but at sub-therapeutic levels, while the liver-targeted study raised expression to therapeutic levels at the highest dose, although duration of expression was only short-lived (<10 weeks). Moreover, the high prevalence of hepatitis in the adult hemophilia population limits the utility of liver as a target tissue in practice. Therefore we investigated the possibility of targeting other cell types. Endothelial cells (ECs) are of interest because of their easy accessibility and the direct secretion of transgene product into the circulation.

The DNA minor groove binding agents Hoechst 33258 and 33342 enhance recombinant adeno‐associated virus (rAAV) transgene expression

Recombinant adeno-associated viruses (rAAV) are commonly used in pre-clinical and clinical gene transfer studies. However, the relatively slow kinetics of rAAV transgene expression complicates in vitro and in vivo experiments. 293 and COS-1 cells were transduced with rAAV2-EGFP, rAAV1-EGFP, or rAAV5-EGFP. The rAAV-EGFP expression was analyzed in the presence of Hoechst 33 258 or 33 342 as a function of time and concentration by flow cytometry and fluorescent microscope. Effects of Hoechst on cell cycle populations were determined by flow cytometry. Enhanced green fluorescent protein (EGFP) expression plasmids with or without AAV inverted terminal repeats (ITR) were constructed and gene expression by transient transfection was compared in the presence of Hoechst. We found that Hoechst 33 258 and 33 342 increase both the level and the population of EGFP gene expressing cells, transduced by several different serotypes of rAAV-EGFP. The augmentation of rAAV-EGFP expression occurs in different cell types in a concentration-dependent manner. In addition, the Hoechst 33 258 or 33 342 mediated enhancement of rAAV gene expression correlated with an increase of cells in S phase and G2/M phases of the cell cycle. Finally, gene expression from transfected ITR-containing plasmid DNA was also enhanced by Hoechst dyes. Our results revealed that two different, although related, DNA-binding drugs, Hoechst 33 258 and 33 342, accelerate the kinetics of rAAV transgene expression. These findings may provide the basis for more sensitive assessment of rAAV biological activity and also extend the applications of rAAV for in vivo gene transfer.

Clinical gene transfer studies for hemophilia A.

The recent advances in gene transfer technology have expedited the development of gene therapy for the treatment of hemophilia A. Three different U.S. Food and Drug Administration-approved phase I clinical trials had been initiated using different gene therapy approaches each with their own advantages and limitations. In the first gene therapy trial for hemophilia A, a non-viral approach was being explored for patients with severe hemophilia A using ex vivo transfected dermal fibroblast expressing B-domain-deleted factor VIII ( BDD-FVIII). There were no serious adverse events and some patients appeared to have experienced fewer bleeding episodes with very low levels of FVIII near baseline. In the second trial, onco-retroviral vectors expressing BDD-FVIII were injected by peripheral intravenous infusion in adult patients suffering from severe hemophilia A. The procedure was safe and in some patients FVIII-transduced cells were detectable in the peripheral blood for more than a year. Although no sustained FVIII expression was detectable, occasional modest changes in FVIII levels were apparent, and in some cases a reduced bleeding frequency occurred compared with historical rates. In another trial, one patient suffering from severe hemophilia A has been treated with a high-capacity (or gutless) adenoviral vector expressing full-length FVIII, which appeared to have resulted in 1% of normal FVIII levels for several months. However, a transient inflammatory response with hematologic and liver abnormalities was observed. In conclusion, although modest improvements in clinical end points have been detected in some patients in these early phase I trials, further improvements in gene delivery technologies are warranted to bring hemophilia A gene therapy one step closer to reality.

759. The DNA Minor Groove Binding Agents Hoechst 33258 and 33342 Enhance Recombinant Adeno-Associated Virus (rAAV) Transgene Expression

Recombinant adeno-associated viruses (rAAV) are commonly used in pre-clinical and clinical gene transfer studies. The relatively slow kinetics of rAAV transgene expression, however, complicates in vitro and in vivo experiments. Accelerating the kinetics of rAAV transgene expression would extend the applications of rAAV for gene transfer and facilitate the determination of vector biological activity in vitro. We found that the DNA minor groove binding agents, Hoechst 33258 and 33342, enhance rAAV gene expression. Hoechst 33258 or 33342 increase both the level of reporter gene expression and the population of transgene expressing cells. We found that Hoechst 33258 and 33342 augment rAAV gene expression in different cell types and in a concentration dependent manner. Hoechst 33258 or 33342 enhanced gene expression for rAAV-1, rAAV-2 and rAAV-5. Hoechst 33258 and 33342 mediated enhancement of rAAV gene expression correlated with an increase in S phase and G2/M phases of the cell cycle. Our data revealed that two different, although related, DNA binding drugs effect rAAV gene expression in a similar manner, suggesting that a common pathway is involved. Our results suggest that Hoechst 33258 and 33342 are potential enhancer drugs for the gene expression and might be useful for gene therapy applications.

85. High-Level Expression of Wild-Type p53 from the Novel p53-Responsive pCLPG Retrovirus Halts the Proliferation of Tumor Cells Regardless of Their p53 Status

One of the barriers to the success of gene therapy is appropriate and adequate expression of the therapeutic gene by the vector. Although retroviral vectors have been widely used in both laboratory and clinical gene transfer studies, many reports in the literature indicate that retroviral vectors suffer silencing of transgene expression in vivo and that modification of the LTR is necessary in order to restore reliable transcription. To this end, we have constructed a retroviral gene transfer system with a modified LTR such that transactivation is mediated by the tumor suppressor p53. Our approach may be beneficial for the achievement of high-level transgene expression as well as inducible transactivation. Ex vivo applications of the pCLPG system would rely on endogenous p53 to drive viral expression implying that stimulation of p53 would result in increased transgene levels (Figure 1A). This transcriptional mechanism, in combination with drug resistance genes, may prove beneficial for the protection of the hematopoietic system from the effects of chemotherapy. In situ applications may benefit from the positive feedback mechanism established by the combination of the p53-responsive LTR plus virally encoded p53, resulting in high-level p53 expression and subsequent suppression of tumor cell proliferation (Figure 1B).

Safety Considerations Associated with Development and Clinical Application of Lentiviral Vector Systems for Gene Transfer

Lentiviral vector systems continue to be developed and investigated for possible use in clinical gene transfer studies and for application as potential gene therapies. These vector systems, as a new technology and potentially novel medical therapy, raise a number of safety concerns, both real and perceived. Careful and thorough characterization of the lentiviral vector systems and of how they might interact with cellular genomes and other pathogens is necessary in order to maximize the safety of this technology as it is developed and the safety of both individuals undergoing gene transfer procedures and the general population. Identification of potential safety concerns should not prevent or unnecessarily delay the development of lentiviral vectors for use in clinical gene transfer, but suggest areas of investigation where it would be wise to allocate resources and effort. Keywords: gene therapy, lentivirus, viral vector, safety, recombination, insertional mutagenesis, human endogenous retrovirus (herv), human immunodeficiency virus (hiv)

Adenosine deaminase gene therapy protocol revisited.

Adenosine deaminase gene therapy protocol revisited.

Clinical gene transfer: education is the key.

Profound damage to public confidence in the discipline of gene therapy has been done in the past two years. The gene transfer incident at the University of Pennsylvania in 1999 and alleged financial conflicts associated with that trial, as well as subsequent revelations of massive underreporting of patient adverse events to the Recombinant DNA Advisory Committee (RAC) as required by the NIH Guidelines on Recombinant DNA Research, combined to cause much of this damage. In response to public concerns and congressional inquiries, relevant federal agencies, including the Food and Drug Administration (FDA), the Office of Biotechnology Activities (OBA) at the NIH, and the Department of Human and Health Services (DHHS), have tightened existing regulations and issued new rules and guidelines governing clinical gene transfer studies. One common feature among all of these federal activities is a call for education of investigators and team members to make them thoroughly familiar with all aspects of clinical gene transfer studies and to better protect patient safety. In response to this challenge, the ASGT sponsored a comprehensive Clinical Gene Transfer Training Course over a two-day period immediately preceding the Society’s 4th Annual Meeting in Seattle this past June. The training course was co-organized and cosponsored by the FDA, OBA, OHRP, and the Mount Sinai School of Medicine, and supported financially by only the OBA and the ASGT. Topics encompassed the following areas: 1. Planning for a Clinical Trial; 2. Preclinical Development of Gene Transfer Vectors; 3. Clinical Trial Design, Approval Process, and Trial Conduct; 4. Clinical Trial Compliance, Monitoring, and Oversight; and 5. Bioethics, Research Integrity, and Conflicts of Interest. Training course objectives were to provide education to basic scientists and trainees on the principles of Good Laboratory Practice (GLP) in generating and analyzing preclinical efficacy and pharmacology/toxicity data to support clinical translational research; production facility directors and staff members on the principles of Good Manufacturing Practice (GMP) in manufacturing and testing of gene transfer products for human applications; and clinical investigators and team members on the principles of Good Clinical Practices (GCP) in accordance with the International Conference on Harmonization (ICH) in conducting clinical trials. In addition, all registrants were educated on the relevant federal regulations and guidelines governing clinical gene transfer studies.

Cystic fibrosis sputum: a barrier to the transport of nanospheres.

Cystic fibrosis (CF) is characterized by the presence of a viscoelastic mucus layer in the upper airways and bronchi. The underlying problem is a mutation in the gene encoding the cystic fibrosis transmembrane conductance regulator protein. Clinical studies of gene transfer for CF are ongoing. For gene delivery to the airways of CF patients to be effective, the mucus covering the target cells must be overcome. We therefore examined the extent to which CF sputum presents a physical barrier to the transport of nanospheres of a size comparable to that of lipoplexes and other transfection systems currently being clinically evaluated for CF gene therapy. We observed that an extremely low percentage of nanospheres (< 0.3%) moved through a 220-microm-thick CF sputum layer after 150 min. The largest nanospheres studied (560 nm) were almost completely blocked by the sputum, whereas the smaller nanospheres (124 nm) were retarded only by a factor of 1.3 as compared with buffer. Surprisingly, the nanospheres diffused significantly more easily through the more viscoelastic sputum samples. We hypothesize that the structure of the network in sputum becomes more macroporous when the sputum becomes more viscoelastic. Sputum from a patient with chronic obstructive pulmonary disease retarded the transport of nanospheres to the same extent as did CF sputum. When directly mixed with CF sputum, recombinant human deoxyribonuclease I moderately facilitated the transport of nanospheres through CF sputum.

From the ASGT president

From the ASGT president

Gene therapy and pancreatic cancer.

Adenocarcinoma of the pancreas is associated with a short survival due to frequent delays in diagnosis and the lack of effective systemic therapies. Advances in understanding the molecular basis of pancreatic cancer have allowed identification of molecular targets which are amenable to therapeutic intervention. Such targets include p53, K-ras, p16, and DPC-4. Gene therapy involves the transfer of genetic constructs which alter the neoplastic potential of the cancer cell. Vectors used in gene transfer include viral and non-viral methods. Presently, gene therapy of pancreatic cancer is limited to pre-clinical studies using in vitro and in vivo models. However, the initial results from these pre-clinical studies have been encouraging and will form the basis for clinical studies of gene transfer in patients with pancreatic cancer.

Human gene therapy in gastrointestinal diseases: in vivo and in vitro approaches.

The first clinical trial in human gene therapy began in 1989 with the successful introduction of marker genes into peripheral blood cells as tumor-infiltrating lymphocytes (TIL) in order to investigate the biological behavior of manipulated cells in humans. In further studies, it was possible to ameliorate clinical genetic diseases based on only one single genetic defect such as adenosine deaminase deficiency (ADA) by repeated infusion of manipulated peripheral blood cells. Meanwhile, a multitude of clinical gene transfer studies were initiated. Three main strategies have thus far been applied in human cancer gene therapy: (1) Reinforcement of the body's immune response by gene transfer into immunological cells; (2) reinforcement of the immune response by manipulating tumor cells; and (3) transfer of drug-sensitive genes into tumor cells with subsequent drug treatment. The first clinical trial in gene therapy for gastrointestinal diseases was performed in 1992 with the introduction of the low-density protein receptor gene (LDL) into liver tissue. Human cancer gene therapy of gastrointestinal diseases is still only in the initial phase of research.

Genetic Marking and Manipulation of Hematopoietic Progenitor Cells Using Retroviral Vectors

In the 5 years since the first human gene therapy studies began, more than 70 clinical protocols have been approved, and over 200 patients have received genetically modified cells. A high proportion of these protocols have made use of hematopoietic progenitor cells or their mature progeny. In this review, we discuss the progress and limitations of current clinical gene transfer studies using marrow-derived progenitor cells and describe how the technique is being applied to the treatment of single-gene disorders and to augment cancer immunotherapies. We also discuss the contribution made by gene marking studies.

An Approach for the Analysis of Relapse and Marrow Reconstitution After Autologous Marrow Transplantation Using Retrovirus-Mediated Gene Transfer

Autologous bone marrow transplantation (ABMT) is widely used as treatment for malignant disease. Although the major cause of treatment failure is relapse, it is unknown if this arises entirely because of residual disease in the patient or whether contaminating cells in the rescuing marrow contribute. Attempts to purge marrow of its putative residual malignant cells may delay hematopoietic reconstitution and are of uncertain efficacy. We now describe how retrovirus-mediated gene transfer may be used to elucidate the source of relapse after ABMT for acute myeloid leukemia and to evaluate the efficacy of purging. Clonogenic myeloid leukemic blast cells in patient marrow can be transduced with the NeoR gene-containing helper-free retrovirus, LNL6, with an efficacy of 0% to 23.5% (mean, 10.5%). Transduced colonies grow in selective media and the presence of the marker gene can be confirmed in individual malignant colonies by polymerase chain reaction. If such malignant cells remain in harvested "remission" marrow, they will therefore be marked after exposure to LNL6. Detection of the marker gene in the malignant cells present at any later relapse would be firm evidence that residual disease contributed to disease recurrence, and would permit rapid subsequent evaluation of purging techniques. The technique also marks normal marrow progenitors from patients with acute myeloblastic leukemia. These colony-forming cells can be detected in long-term marrow cultures at a frequency of 1% to 18% for up to 10 weeks after exposure to the vector. Animal models and analysis of probability tables both suggest that these levels of marking in vitro are sufficient to provide information about the mechanisms of relapse and the biology of marrow regeneration in vivo. These preclinical data form part of the basis for current clinical studies of gene transfer into marrow before ABMT.

An approach for the analysis of relapse and marrow reconstitution after autologous marrow transplantation using retrovirus-mediated gene transfer

Autologous bone marrow transplantation (ABMT) is widely used as treatment for malignant disease. Although the major cause of treatment failure is relapse, it is unknown if this arises entirely because of residual disease in the patient or whether contaminating cells in the rescuing marrow contribute. Attempts to purge marrow of its putative residual malignant cells may delay hematopoietic reconstitution and are of uncertain efficacy. We now describe how retrovirus-mediated gene transfer may be used to elucidate the source of relapse after ABMT for acute myeloid leukemia and to evaluate the efficacy of purging. Clonogenic myeloid leukemic blast cells in patient marrow can be transduced with the NeoR gene-containing helper-free retrovirus, LNL6, with an efficacy of 0% to 23.5% (mean, 10.5%). Transduced colonies grow in selective media and the presence of the marker gene can be confirmed in individual malignant colonies by polymerase chain reaction. If such malignant cells remain in harvested “remission” marrow, they will therefore be marked after exposure to LNL6. Detection of the marker gene in the malignant cells present at any later relapse would be firm evidence that residual disease contributed to disease recurrence, and would permit rapid subsequent evaluation of purging techniques. The technique also marks normal marrow progenitors from patients with acute myeloblastic leukemia. These colony-forming cells can be detected in long-term marrow cultures at a frequency of 1% to 18% for up to 10 weeks after exposure to the vector. Animal models and analysis of probability tables both suggest that these levels of marking in vitro are sufficient to provide information about the mechanisms of relapse and the biology of marrow regeneration in vivo. These preclinical data form part of the basis for current clinical studies of gene transfer into marrow before ABMT.