Gene delivery is the process of introducing foreign genetic material, such as DNA or

RNA Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...

, into host

cells Cell most often refers to: * Cell (biology), the functional basic unit of life Cell may also refer to: Locations * Monastic cell, a small room, hut, or cave in which a religious recluse lives, alternatively the small precursor of a monastery w ...

. Gene delivery must reach the

genome In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ...

of the host cell to induce

gene expression Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, protein or non-coding RNA, and ultimately affect a phenotype, as the final effect. T ...

. Successful gene delivery requires the foreign gene delivery to remain stable within the host cell and can either integrate into the

or replicate independently of it. This requires foreign DNA to be synthesized as part of a

vector Vector most often refers to: *Euclidean vector, a quantity with a magnitude and a direction *Vector (epidemiology), an agent that carries and transmits an infectious pathogen into another living organism Vector may also refer to: Mathematic ...

, which is designed to enter the desired host cell and deliver the

transgene A transgene is a gene that has been transferred naturally, or by any of a number of genetic engineering techniques, from one organism to another. The introduction of a transgene, in a process known as transgenesis, has the potential to change the ...

to that cell's genome. Vectors utilized as the method for gene delivery can be divided into two categories, recombinant viruses and synthetic vectors (viral and non-viral). In complex multicellular

eukaryote Eukaryotes () are organisms whose cells have a nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the three domains of life. Bact ...

s (more specifically Weissmanists), if the

is incorporated into the host's

germline In biology and genetics, the germline is the population of a multicellular organism's cells that pass on their genetic material to the progeny ( offspring). In other words, they are the cells that form the egg, sperm and the fertilised egg. T ...

cells, the resulting host cell can pass the transgene to its progeny. If the transgene is incorporated into somatic cells, the transgene will stay with the somatic cell line, and thus its host organism. Gene delivery is a necessary step in

gene therapy Gene therapy is a medical field which focuses on the genetic modification of cells to produce a therapeutic effect or the treatment of disease by repairing or reconstructing defective genetic material. The first attempt at modifying human DN ...

for the introduction or silencing of a gene to promote a therapeutic outcome in patients and also has applications in the genetic modification of crops. There are many different methods of gene delivery for various types of cells and tissues.

History

Viral based vectors emerged in the 1980s as a tool for transgene expression. In 1983, Albert Siegel described the use of viral vectors in plant transgene expression although viral manipulation via cDNA cloning was not yet available. The first virus to be used as a vaccine vector was the

vaccinia ''Vaccinia virus'' (VACV or VV) is a large, complex, enveloped virus belonging to the poxvirus family. It has a linear, double-stranded DNA genome approximately 190 kbp in length, which encodes approximately 250 genes. The dimensions of the ...

virus in 1984 as a way to protect chimpanzees against

hepatitis B Hepatitis B is an infectious disease caused by the ''Hepatitis B virus'' (HBV) that affects the liver; it is a type of viral hepatitis. It can cause both acute and chronic infection. Many people have no symptoms during an initial infection. For ...

. Non-viral gene delivery was first reported on in 1943 by Avery et al. who showed cellular phenotype change via

exogenous DNA Exogenous DNA is DNA originating outside the organism of concern or study. Exogenous DNA can be found naturally in the form of partially degraded fragments left over from dead cells. These DNA fragments may then become integrated into the chromos ...

exposure.

Methods

There are a variety of methods available to deliver genes to host cells. When genes are delivered to bacteria or plants the process is called transformation and when it is used to deliver genes to animals it is called

transfection Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. It may also refer to other methods and cell types, although other terms are often preferred: " transformation" is typically used to des ...

. This is because transformation has a different meaning in relation to animals, indicating progression to a cancerous state. For some bacteria no external methods are need to introduce genes as they are naturally able to take up foreign DNA. Most cells require some sort of intervention to make the cell membrane permeable to DNA and allow the DNA to be stably inserted into the hosts

Chemical

Chemical based methods of gene delivery can use natural or synthetic compounds to form particles that facilitate the transfer of genes into cells. These synthetic vectors have the ability to electrostatically bind DNA or RNA and compact the genetic information to accommodate larger genetic transfers. Chemical vectors usually enter cells by

endocytosis Endocytosis is a cellular process in which substances are brought into the cell. The material to be internalized is surrounded by an area of cell membrane, which then buds off inside the cell to form a vesicle containing the ingested material. E ...

and can protect genetic material from degradation.

Heat shock

One of the simplest method involves altering the environment of the cell and then stressing it by giving it a heat shock. Typically the cells are incubated in a solution containing

divalent In chemistry, the valence (US spelling) or valency (British spelling) of an element is the measure of its combining capacity with other atoms when it forms chemical compounds or molecules. Description The combining capacity, or affinity of an ...

cation An ion () is an atom or molecule with a net electrical charge. The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by convent ...

s (often

calcium chloride Calcium chloride is an inorganic compound, a salt with the chemical formula . It is a white crystalline solid at room temperature, and it is highly soluble in water. It can be created by neutralising hydrochloric acid with calcium hydroxide. C ...

) under cold conditions, before being exposed to a heat pulse. Calcium chloride partially disrupts the cell membrane, which allows the recombinant DNA to enter the host cell. It is suggested that exposing the cells to divalent cations in cold condition may change or weaken the cell surface structure, making it more permeable to DNA. The heat-pulse is thought to create a thermal imbalance across the cell membrane, which forces the DNA to enter the cells through either cell pores or the damaged cell wall.

Calcium phosphate

Another simple methods involves using

calcium phosphate The term calcium phosphate refers to a family of materials and minerals containing calcium ions (Ca2+) together with inorganic phosphate anions. Some so-called calcium phosphates contain oxide and hydroxide as well. Calcium phosphates are w ...

to bind the DNA and then exposing it to cultured cells. The solution, along with the DNA, is encapsulated by the cells and a small amount of DNA can be integrated into the genome.

Liposomes and polymers

Liposome A liposome is a small artificial vesicle, spherical in shape, having at least one lipid bilayer. Due to their hydrophobicity and/or hydrophilicity, biocompatibility, particle size and many other properties, liposomes can be used as drug deliver ...

s and

polymer A polymer (; Greek '' poly-'', "many" + '' -mer'', "part") is a substance or material consisting of very large molecules called macromolecules, composed of many repeating subunits. Due to their broad spectrum of properties, both synthetic a ...

s can be used as vectors to deliver DNA into cells. Positively charged liposomes bind with the negatively charged DNA, while polymers can be designed that interact with DNA. They form lipoplexes and polyplexes respectively, which are then up-taken by the cells. The two systems can also be combined. Polymer-based non-viral vectors uses polymers to interact with DNA and form polyplexes.

Nanoparticles

The use of engineered inorganic and organic

nanoparticle A nanoparticle or ultrafine particle is usually defined as a particle of matter that is between 1 and 100 nanometres (nm) in diameter. The term is sometimes used for larger particles, up to 500 nm, or fibers and tubes that are less than 1 ...

s is another non-viral approach for gene delivery.

Physical

Artificial gene delivery can be mediated by physical methods which uses force to introduce genetic material through the cell membrane.

Electroporation

Electroporation Electroporation, or electropermeabilization, is a microbiology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing chemicals, drugs, electrode arrays or DNA to be introdu ...

is a method of promoting competence. Cells are briefly shocked with an

electric field An electric field (sometimes E-field) is the physical field that surrounds electrically charged particles and exerts force on all other charged particles in the field, either attracting or repelling them. It also refers to the physical field ...

of 10-20 kV/cm, which is thought to create holes in the cell membrane through which the plasmid DNA may enter. After the electric shock, the holes are rapidly closed by the cell's membrane-repair mechanisms.

Biolistics

Another method used to transform plant cells is biolistics, where particles of gold or tungsten are coated with DNA and then shot into young plant cells or plant embryos. Some genetic material enters the cells and transforms them. This method can be used on plants that are not susceptible to ''Agrobacterium'' infection and also allows transformation of plant

plastids The plastid (Greek: πλαστός; plastós: formed, molded – plural plastids) is a membrane-bound organelle found in the cells of plants, algae, and some other eukaryotic organisms. They are considered to be intracellular endosymbiotic cyanob ...

. Plants cells can also be transformed using electroporation, which uses an electric shock to make the cell membrane permeable to plasmid DNA. Due to the damage caused to the cells and DNA the transformation efficiency of biolistics and electroporation is lower than agrobacterial transformation.

Microinjection

Microinjection Microinjection is the use of a glass micropipette to inject a liquid substance at a microscopic or borderline macroscopic level. The target is often a living cell but may also include intercellular space. Microinjection is a simple mechanical pr ...

is where DNA is injected through the cell's

nuclear envelope The nuclear envelope, also known as the nuclear membrane, is made up of two lipid bilayer membranes that in eukaryotic cells surround the nucleus, which encloses the genetic material. The nuclear envelope consists of two lipid bilayer membr ...

directly into the

nucleus Nucleus ( : nuclei) is a Latin word for the seed inside a fruit. It most often refers to: *Atomic nucleus, the very dense central region of an atom *Cell nucleus, a central organelle of a eukaryotic cell, containing most of the cell's DNA Nucle ...

Sonoporation

Sonoporation is the transient permeation of

cell membranes The cell membrane (also known as the plasma membrane (PM) or cytoplasmic membrane, and historically referred to as the plasmalemma) is a biological membrane that separates and protects the interior of all cells from the outside environment (the ...

assisted by

ultrasound Ultrasound is sound waves with frequencies higher than the upper audible limit of human hearing. Ultrasound is not different from "normal" (audible) sound in its physical properties, except that humans cannot hear it. This limit varies ...

, typically in the presence of gas

microbubbles Microbubbles (MBs) are bubbles smaller than one hundredth of a millimetre in diameter, but larger than one micrometre. They have widespread application in industry, life science, and medicine. The composition of the bubble shell and filling materia ...

. Sonoporation allows for the entry of genetic material into cells.

Photoporation

Photoporation is when laser pulses are used to create pores in a cell membrane to allow entry of genetic material.

Magnetofection

Magnetofection Magnetofection is a transfection method that uses magnetic fields to concentrate particles containing vectors to target cells in the body. Magnetofection has been adapted to a variety of vectors, including nucleic acids, non-viral transfection sys ...

uses magnetic particles complexed with DNA and an external magnetic field concentrate nucleic acid particles into target cells.

Hydroporation

A hydrodynamic capillary effect can be used to manipulate cell permeability.

Agrobacterium

In plants the DNA is often inserted using ''Agrobacterium''-mediated recombination, taking advantage of the ''Agrobacterium''s T-DNA sequence that allows natural insertion of genetic material into plant cells. Plant tissue are cut into small pieces and soaked in a fluid containing suspended ''Agrobacterium''. The bacteria will attach to many of the plant cells exposed by the cuts. The bacteria uses

conjugation Conjugation or conjugate may refer to: Linguistics *Grammatical conjugation, the modification of a verb from its basic form * Emotive conjugation or Russell's conjugation, the use of loaded language Mathematics *Complex conjugation, the change ...

to transfer a DNA segment called T-DNA from its plasmid into the plant. The transferred DNA is piloted to the plant cell nucleus and integrated into the host plants genomic DNA.The plasmid T-DNA is integrated semi-randomly into the

of the host cell. By modifying the plasmid to express the gene of interest, researchers can insert their chosen gene stably into the plants genome. The only essential parts of the T-DNA are its two small (25 base pair) border repeats, at least one of which is needed for plant transformation. The genes to be introduced into the plant are cloned into a plant transformation vector that contains the T-DNA region of the

plasmid A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; howe ...

. An alternative method is

agroinfiltration Agroinfiltration is a method used in plant biology and especially lately in plant biotechnology to induce transient expression of genes in a plant, or isolated leaves from a plant, or even in cultures of plant cells, in order to produce a desire ...

Viral delivery

Virus A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria and archaea. Since Dmitri Ivanovsky' ...

mediated gene delivery utilizes the ability of a virus to inject its DNA inside a host cell and takes advantage of the virus' own ability to replicate and implement their own genetic material. Viral methods of gene delivery are more likely to induce an immune response, but they have high efficiency. Transduction is the process that describes virus-mediated insertion of DNA into the host cell. Viruses are a particularly effective form of gene delivery because the structure of the virus prevents degradation via

lysosome A lysosome () is a membrane-bound organelle found in many animal cells. They are spherical vesicles that contain hydrolytic enzymes that can break down many kinds of biomolecules. A lysosome has a specific composition, of both its membrane p ...

s of the DNA it is delivering to the nucleus of the host cell. In gene therapy a gene that is intended for delivery is packaged into a replication-deficient viral particle to form a

viral vector Viral vectors are tools commonly used by molecular biologists to deliver genetic material into cells. This process can be performed inside a living organism (''in vivo'') or in cell culture (''in vitro''). Viruses have evolved specialized molecu ...

. Viruses used for gene therapy to date include retrovirus, adenovirus, adeno-associated virus and herpes simplex virus. However, there are drawbacks to using viruses to deliver genes into cells. Viruses can only deliver very small pieces of DNA into the cells, it is labor-intensive and there are risks of random insertion sites, cytopathic effects and mutagenesis. Viral vector based gene delivery uses a

to deliver genetic material to the host cell. This is done by using a virus that contains the desired gene and removing the part of the viruses genome that is infectious. Viruses are efficient at delivering genetic material to the host cell's nucleus, which is vital for replication.

RNA-based viral vectors

RNA-based viruses were developed because of the ability to transcribe directly from infectious RNA transcripts. RNA vectors are quickly expressed and expressed in the targeted form since no processing is required ource needed Retroviral vectors include oncoretroviral,

lentiviral ''Lentivirus'' is a genus of retroviruses that cause chronic and deadly diseases characterized by long incubation periods, in humans and other mammalian species. The genus includes the human immunodeficiency virus (HIV), which causes AIDS. Lent ...

and human foamy virus are RNA-based viral vectors that reverse transcript and integrated into the host genome, permits long-term transgene expression .

DNA-based viral vectors

DNA-based viral vectors include

Adenoviridae Adenoviruses (members of the family ''Adenoviridae'') are medium-sized (90–100 nm), nonenveloped (without an outer lipid bilayer) viruses with an icosahedral nucleocapsid containing a double-stranded DNA genome. Their name derives from ...

adeno-associated virus Adeno-associated viruses (AAV) are small viruses that infect humans and some other primate species. They belong to the genus '' Dependoparvovirus'', which in turn belongs to the family ''Parvoviridae''. They are small (approximately 26 nm in d ...

and

herpes simplex virus Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), also known by their taxonomical names '' Human alphaherpesvirus 1'' and '' Human alphaherpesvirus 2'', are two members of the human ''Herpesviridae'' family, a set of viruses that produce viral in ...

Applications

Gene therapy

Several of the methods used to facilitate gene delivery have applications for therapeutic purposes.

Gene therapy Gene therapy is a medical field which focuses on the genetic modification of cells to produce a therapeutic effect or the treatment of disease by repairing or reconstructing defective genetic material. The first attempt at modifying human DN ...

utilizes gene delivery to deliver genetic material with the goal of treating a disease or condition in the cell. Gene delivery in therapeutic settings utilizes non-

immunogenic Immunogenicity is the ability of a foreign substance, such as an antigen, to provoke an immune response in the body of a human or other animal. It may be wanted or unwanted: * Wanted immunogenicity typically relates to vaccines, where the injectio ...

vectors capable of cell specificity that can deliver an adequate amount of transgene expression to cause the desired effect. Advances in

genomics Genomics is an interdisciplinary field of biology focusing on the structure, function, evolution, mapping, and editing of genomes. A genome is an organism's complete set of DNA, including all of its genes as well as its hierarchical, three-dim ...

have enabled a variety of new methods and gene targets to be identified for possible applications.

DNA microarray A DNA microarray (also commonly known as DNA chip or biochip) is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to ...

s used in a variety of

next-gen sequencing DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. The ...

can identify thousands of genes simultaneously, with analytical software looking at gene expression patterns, and orthologou s genes in model species to identify function. This has allowed a variety of possible vectors to be identified for use in gene therapy. As a method for creating a new class of vaccine, gene delivery has been utilized to generate a

hybrid Hybrid may refer to: Science * Hybrid (biology), an offspring resulting from cross-breeding ** Hybrid grape, grape varieties produced by cross-breeding two ''Vitis'' species ** Hybridity, the property of a hybrid plant which is a union of two dif ...

biosynthetic vector to deliver a possible vaccine. This vector overcomes traditional barriers to gene delivery by combining ''E. coli'' with a

synthetic polymer Some familiar household synthetic polymers include: Nylons in textiles and fabrics, Teflon in non-stick pans, Bakelite for electrical switches, polyvinyl chloride (PVC) in pipes, etc. The common PET bottles are made of a synthetic polymer, pol ...

to create a vector that maintains plasmid DNA while having an increased ability to avoid degradation by target cell lysosomes.

References

External links

The 10th US-Japan Symposium on Drug Delivery SystemsNature: Gene DeliveryGenetic Science Learning Center: Gene DeliveryGenome EditingNIH: How does gene therapy work?
Applied genetics Biotechnology