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