Vol. 52, Issue 4, 493-512, December 2000

Design of Retroviral Vectors and Helper Cells for Gene Therapy

Wei-Shau Hu¹ and Vinay K. Pathak

Department of Microbiology and Immunology (W.-S.H.), Department of Biochemistry (V.K.P.), Mary Babb Randolph Cancer Center (W.-S.H., V.K.P.), School of Medicine, West Virginia University, Morgantown, West Virginia; and HIV Drug Resistance Program, National Cancer Institute, FCRDC, Frederick, Maryland (W.-S.H., V.K.P.)

I. General Introduction and Scope
II. Background
    A. Replication Cycle of Retroviruses
    B. Genome Structure of Retroviruses
III. Basic Concepts in Retrovirus Vectors and Helper Cells
IV. Helper Cells and Packaging Systems
    A. Helper Cell Lines
    B. One-Genome Helper Constructs
    C. Split-Genome Helper Constructs
    D. Inducible Helper Constructs
    E. Transient Transfection Systems
    F. Systems That Generate Pseudotyped Viruses
    G. Systems Containing Genetically Modified env for Cell or Tissue Targeting
    H. Hybrid Systems
V. Vectors Based on Different Retroviruses
    A. Vectors Derived from Oncoviruses
        1. Murine Leukemia Virus-Based Vectors.
        2. Spleen Necrosis Virus-Based Vectors.
        3. Rous Sarcoma Virus- and Avian Leukosis Virus-Based Vectors.
    B. Vectors Derived from Lentiviruses
    C. Vectors Derived from Spumaviruses
VI. Design of Retroviral Vectors
    A. Standard Vectors
        1. U3 Promoter-Driven Gene Expression.
        2. Vectors That Use an Internal Promoter to Express Additional Genes.
        3. Vectors That Use Splicing to Express Additional Genes.
        4. Vectors That Use Translational Control Signals to Express Additional Genes.
    B. Double-Copy Vectors
    C. Self-Inactivating Vectors
        1. U3 Minus Vectors.
        2. Cre/loxP Vectors.
    D. Self-Inactivating and Self-Activating Vectors
    E. Vectors Targeted to Specific Cells
    F. Vectors That Utilize Cell-Type-Specific Promoters
VII. General Considerations for Using Retrovirus Vectors and Helper Cells
    A. Gene Expression
    B. Virus Host Range and Titers
        1. Considerations for Envelope Selection and Virus Host Range.
        2. Ping-Pong Amplification.
        3. Concentration of Viruses.
    C. Safety Concerns Associated with Retrovirus-Based Gene Therapy
VIII. Gene Therapy Applications and Future Directions
    A. Retroviral Vectors Used in Gene Therapy Clinical Trials
    B. Future Directions
Acknowledgments
References

During the past decade, gene therapy has been applied to the treatment of disease in hundreds of clinical trials. Various tools have been developed to deliver genes into human cells; among them, genetically engineered retroviruses are currently the most popular tool for gene delivery. Most of the systems contain vectors that are capable of accommodating genes of interest and helper cells that can provide the viral structural proteins and enzymes to allow for the generation of vector-containing infectious viral particles. Retroviridae is a family of retroviruses that differs in nucleotide and amino acid sequence, genome structure, pathogenicity, and host range. This diversity provides opportunities to use viruses with different biological characteristics to develop different therapeutic applications. Currently, a variety of retroviruses that provide distinct advantages for gene delivery has been modified and used in clinical trials. In this review, the genome structures of oncoviruses, lentiviruses, and spumaviruses are reviewed and examples of vectors derived from these viruses are described. As with any delivery tool, the efficiency, the ability to target certain tissue or cell type, the expression of the gene of interest, and the safety of retroviral-based systems are important for successful application of gene therapy. Significant efforts have been dedicated to these areas of research in recent years. Various modifications have been made to retroviral-based vectors and helper cells to alter gene expression, target delivery, improve viral titers, and increase safety. The principles and design of these modifications are discussed in this review.