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    Vectors For Gene Delivery

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    Muhammad Ikram Rabbani
    There are several methods for delivering genes into cells, including viral and non-viral methods. Viral vectors such as retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses can efficiently transduce cells, but have limitations such as immunogenicity, inability to transduce non-dividing cells, and short duration of transgene expression. Non-viral methods including chemical methods like calcium phosphate and lipid-based transfection, as well as physical methods like electroporation and gene guns, have advantages over viral vectors in that they do not cause immune responses but have lower transduction efficiencies. The development of novel formulations aims to enhance the delivery and expression of therapeutic genes using non-

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    Vectors For Gene Delivery

    Uploaded by

    Muhammad Ikram Rabbani
    46 views6 pages
    There are several methods for delivering genes into cells, including viral and non-viral methods. Viral vectors such as retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses can efficiently transduce cells, but have limitations such as immunogenicity, inability to transduce non-dividing cells, and short duration of transgene expression. Non-viral methods including chemical methods like calcium phosphate and lipid-based transfection, as well as physical methods like electroporation and gene guns, have advantages over viral vectors in that they do not cause immune responses but have lower transduction efficiencies. The development of novel formulations aims to enhance the delivery and expression of therapeutic genes using non-

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    Vectors for gene Delivery

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    There are several methods for delivering genes into cells, including viral and non-viral methods. Viral vectors such as retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses can efficiently transduce cells, but have limitations such as immunogenicity, inability to transduce non-dividing cells, and short duration of transgene expression. Non-viral methods including chemical methods like calcium phosphate and lipid-based transfection, as well as physical methods like electroporation and gene guns, have advantages over viral vectors in that they do not cause immune responses but have lower transduction efficiencies. The development of novel formulations aims to enhance the delivery and expression of therapeutic genes using non-

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    46 views6 pages

    Vectors For Gene Delivery

    Uploaded by

    Muhammad Ikram Rabbani
    There are several methods for delivering genes into cells, including viral and non-viral methods. Viral vectors such as retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses can efficiently transduce cells, but have limitations such as immunogenicity, inability to transduce non-dividing cells, and short duration of transgene expression. Non-viral methods including chemical methods like calcium phosphate and lipid-based transfection, as well as physical methods like electroporation and gene guns, have advantages over viral vectors in that they do not cause immune responses but have lower transduction efficiencies. The development of novel formulations aims to enhance the delivery and expression of therapeutic genes using non-

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    © All Rights Reserved
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    Download as pdf or txt
    of 6

    Vectors for gene Delivery

    Genetic material can be transferred via a vector that is defined as the vehicle that is used to deliver the
    gene of interest. The ideal vector would transfer a precise amount of genetic material into each target cell,
    thereby allowing for expression of the gene product without causing toxicity.

    Gene transfer via the viral vectors is called transduction while transfer via the non‑viral vectors is called
    Transfection.

    Gene Transfer via chemical methods


    1. Calcium phosphate
    2. Lipid (liposomes, and lipoplexes)
    3. Protein complexes
    4. DEAE‑dextran for oral gene delivery
    5. Surfactants and perfluro chemical liquids for aerosol delivery of gene

    Physical transfection of Genes


    1. Electroporation
    2. Microinjection

    Diagram of the intracytoplasmic injection of a human cell. Micromanipulator on the left holds the
    cell in position while microinjector on the right delivers the gene of interest.

    3. Gene gun/ Ballistic particles.

    A gene gun is used for injecting cells with genetic information, it is also known as biolistic particle
    delivery system. Gene guns can be used effectively on most cells but are mainly used on plant cells.
    Step 1 The gene gun apparatus is ready to fire. Step 2 When the gun is turned on and the helium
    flows through. Step 3 The helium moving the disk with DNA coated particles toward the screen. Step
    4 The helium having pushed the particles moving through the screen and moving to the target cells to
    transform the cells.

    Methods of gene delivery


    Different methods to deliver therapeutic DNA and proteins to target cells. Non-viral gene delivery methods
    have many advantages over viral vectors in gene therapy. They do not cause immunogenicity and
    carcinogenicity, and can deliver a large size of therapeutic DNA efficiently with a low price tag. As no one-
    size-fits-all solution to therapeutic DNA delivery exits, development, and formulations remain the main focus
    of research on non-viral methods.
    Types of viral vectors

    Integrating (retroviral, lentiviral, and adeno‑associated)


    non‑integrating (adenoviralvector)

    Mechanism of DNA delivery and multiplication of a non-integrating Viral vector.


    1. Retroviral vectors
    RNA viruses
    reverse transcriptase
    DNA intermediate is then incorporated
    e.g murine leukemia virus

    Limitations
    low vector titer,
    low transfection efficiency,
    particle instability
    inability to transduce non‑dividing post‑mitotic cells

    2. Lentiviruses

    Can divide both proliferating and quiescent cells


    Modification of surface glycoproteins to ensure transduction into several cells
    long‑term expression
    Efficient transfer without inflammatory responses

    3. Adenoviral vectors

    DNA viruses
    transfer DNA into the nucleus,
    no integration
    effects are transient (range: 7‑42 days).
    Multiple administrations are required.
    most cells is susceptible to infection, regardless of
    their position in the cell cycle.
    higher titer

    Limitations

    does not integrate its genome into the


    in the nucleus as an episomal element
    90% of individuals have antibodies against these viruses
    In most tissues, the duration of transgene expression is limited to a few days to a week
    viral genes are also transduced and expressed, eliciting an immune response
    early clearance from cells

    Advantages
    ease of purification and concentration and the high efficiency rate of host cell infection
    dividing or non‑dividing cells
    A good candidate for direct in vivo gene transfer.

    Mechanism of adenovirus-mediated delivery of a therapeutic DNA. Upon infection, adenovirus delivers the
    encapsulated therapeutic DNA into the cytoplasm of the target cells. Various stages of viral gene
    delivery, viz cell attachment, internalization, endocytosis, uncoating, transcription and translation of the
    therapeutic protein, are shown.

    4. Adeno Associated Viruses

    Low immunogenicity, no known pathogenicity,


    non‑proliferating cells,
    discrete genome insertion sites.
    “Suicide” gene therapy has been shown to
    cancer models AAV vectors have been shown transduce
    brain, skeletal muscle, liver and possibly CD34 + blood
    cells efficiently.

    Limitations
    High multiplicity of infection (the number of viral particles per cell required to achieve transduction)
    genome is small, only allowing ~4.8 kb of added DNA
    5. herpes simplex virus
    The advantages of the herpes simplex virus (HSV) are its large size can accommodate large pieces of foreign DNA
    The continuous expression of genes from long‑lived infection.
    Little risk of insertional mutagenesis with the HSV
    because it remains outside the nucleus (episomal).
    Ability to remain latent in tissues and to be re‑activated at the original site of infection.
    a common pathogen in humans and rarely causes significant illnesses.

    Limitations
    include low infection efficiency,
    large genome size that makes it more difficult to manipulate than other viral vectors.
    tropism for neural cells limits its action

    6. pox viruses

    The large potential size (25 kb) of the gene insert


    Absence of viral integration in to the host cellular genome,
    immune stimulation induced by this virus
    Vaccinia virus infects all cells; however, the host immune response to the vector does
    not abrogate the tumor immune response even following
    repeated injections.
    The availability of attenuated virus allows the use of vaccinia in immuno‑delicate cancer
    Patients
    vector enhances immunological rejection of the tumor.

    Limitations
    Common toxicities included a local skin reaction at the site of the vaccine
    Mild flu‑like symptoms of 1‑2 days’ duration.
    Cellular immune response did not correlate with the clinical response.

    7. Alpha viruses

    Members of the Togaviridae family and are enveloped, single‑stranded RNA viruses
    There are three different viruses with similar properties useful in gene therapy: Semliki forest virus, Sindbis virus, and
    Venezuelan equine encephalitis virus.
    Most alpha virus vectors used as gene vehicles are replication incompetent.
    They encode only the non‑structural genes, while the structural genes necessary for replication are located in the helper
    plasmid genome.
    To eliminate or at least reduce the possibility of generating infectious replication‑competent particles, structural genes
    can be localized to two different helper plasmids.
    Replication‑competent vectors contain all the genes of the wild‑type genome, including the therapeuticgene, and are
    also able to transduce neighboring and surrounding tumor cells.
    Because of their extensive host‑cell toxicity and broad host range, alpha viruses are suitable for cancer gene therapy
    of various tumors.

    Non-viral Vectors

    1. Bactofection/ bacterial Plasmids


    The use of bacteria as a vector for the delivery of therapeutic genes to target cells is known as bactofection,

    2. DNA vaccination

    It is known that bactofection of plasmids encoding a tumor‑expressed antigens can lead to induction of humoral
    and cellular immune response in the host thereby providing protective defense against tumors.
    This approach, termed DNA vaccination, has been successfully implemented for anti‑angiogenic therapy
    3. Alternative gene therapy

    bacterially expressed therapeutic proteins

    4. Bactoference
    Bacteria that have engineered to produce and deliver short interfering RNA represent a novel tool for the efficient
    induction of RNA interference in host cells.

    Can be divided into two groups


    1. Strictly anaerobic bacteria
    2. Attenuated auxotrophic Strains

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