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Gene Therapy in Clinical Applications

overview: How Do We Translate Gene Therapy to Clinical Trials?

Departments of Oncology and Pediatrics, Johns Hopkins University School of Medicine

Hematopoietic gene therapy is a potentially attractive medical tool because of the biology of the lymphohematopoietic stem cell (HSC). The HSC is a favorable cellular target for permanent introduction of genes into the organism, for several reasons, including:

• Natural and laboratory viruses and potential viral vectors, notably retroviruses, can gain entry to and integrate into the DNA of early hematopoietic progenitor cells.
  
• HSC should provide a lifelong source of amplified progeny expressing the introduced gene.
  
• The multiple lineages of HSC-derived blood and immune cells traverse the entire organism, and so might correct many inherited enzyme deficiency diseases by secreting enzymes (e.g. storage diseases).
  
• Several diseases of blood and immune cells are corrected by successful allogeneic bone marrow transplantation (BMT), and so could potentially be treated by expression of wild type genes in hematopoietic stem cells.
  

Retroviral vectors for hematopoietic gene transfer initially generated many potential concerns, e.g. insertional mutagenesis. Fortunately, most of these concerns have not become clinical problems. Unfortunately, efficacy has been severely limited due to extreme inefficiency of transducing human HSC:

• Transduction of murine in vitro colony-forming progenitors is much more efficient than transduction of murine transplantable HSC.
  
• Transduction of murine in vitro colony-forming progenitors is much more efficient than transduction of human in vitro colony-forming progenitors.
  
• Transduction of human in vitro colony-forming progenitors is much more efficient than transduction of human transplantable HSC.
  

There has been considerable recent progress on increasing the efficiency of retroviral transduction. Is hematopoietic gene therapy now close to clinical trials? If so, what are the next steps toward translating these gains to clinical testing?

Some of the next steps toward translating these gains to clinical testing include:

• Development and utilization of assays that really predict transduction of HSC:
  • Clinical BMT = gold standard
    
  • In vivo chimera assays (human/fetal sheep, human/immunodeficient mouse)
    
  • Novel in vitro surrogates?
    
  
  
• Insuring efficient vector binding to and entry into HSC
  • Pseudotypes; GALV, VSV receptors
    
  
  
• Insuring efficient vector integration into cellular DNA
  • Lentivirus appears to integrate in resting cells
    
  • "Early acting" hematopoietic growth factors appear to recruit HSC into cycle by ex vivo culture before and during transduction with standard RLV
    
  
  
• Avoiding the silencing of successfully transduced therapeutic gene
  • Insulator sequences which block methylation of the therapeutic gene/promoter
    
  
  
• Providing specific enhancers for high expression of the successfully transduced therapeutic gene
  
• Development of systems for regulated expression that would allow on-off switching in the patient
  
• Development of vectors which utilize specific, safe integration sites
  
• Avoiding potentially immunogenic and virally infected human proteins
  • Serum-free medium (added dividend: reproducibility!)
    
  
  

Recently, Enrico Novelli., Linzhao Cheng, Yandan Yang, and I have focused on the use of hematopoietic growth factor stimulation in serum-free medium to address some of the above problems. We have shown that human cord blood CD34⁺ cells can be transduced with retroviral vectors by ex vivo culture in serum-free medium containing the cytokine combination of stem cell factor, thrombopoiesis, and flt-3 ligand. Transduced cells generate easily detectable human hematopoietic engraftment in immunodeficient mice, and a substantial minority of the human marrow cells in the resulting chimeras contain the transduced marker protein. With Dr. Harry Malech, we are now investigating the use of this system for clinical trials.

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