Transfer DNA

TrIP, adapted from the chromatin immuoprecipitation assay, involves formaldehyde treatment of intact cells to cross-link channel subunits to the T-DNA substrate as it exits the cell, disruption of the cells, solubilization of membranes, and immunoprecipitation to recover channel subunits.

From: Encyclopedia of Microbiology (Third Edition) , 2009

Methylation and other Modifications of Nucleic Acids and Proteins

M.G. Marinus , in Encyclopedia of Microbiology (Third Edition), 2009

Restriction and Modification of DNA

DNA transfer of plasmid, bacterial virus or chromosomal DNA is thought to occur at a low level between bacterial strains and species. Most bacteria have surveillance systems that allow them to differentiate 'foreign' DNA from their own DNA. The basis of one of these systems is the possession of an endonuclease (e.g., EcoRI) that recognizes a specific sequence in DNA (5′-GAATTC-3′) and cleaves it only when unmethylated. A corresponding DNA methyltransferase (M.EcoRI) recognizes the identical sequence and modifies it to 5′-GAmeATTC-3′, where the second A is methylated. Therefore, bacteria possessing the EcoRI methyltransferase will have all susceptible sequences methylated and thus be immune to EcoRI endonuclease action. Introduction into the bacterium of homologous or heterologous DNA lacking the specific EcoRI modification leads to recognition and cleavage of such DNA and subsequent degradation by the endonuclease.

A very large number of restriction enzymes have been isolated from bacteria, each having a unique DNA recognition sequence. The availability of this battery of enzymes has been a major contributing factor to the success of recombinant DNA technology in allowing the mapping of genes and their isolation from chromosomes.

One other interesting aspect of restriction/modification is that it can be the basis of epigenetic behavior. For example, bacterial viruses containing DNA not modified at EcoRI sites will not kill bacteria, since their DNA is degraded upon entering the bacteria. However, at a low frequency, a few virus DNA molecules survive because they have been methylated and, when these are tested again, they now infect and kill the same bacteria at high efficiency. The virus has not mutated in any way; the only difference is that its DNA has become methylated and thus is now resistant to EcoRI endonuclease action. Since methylation is dependent on the host in which the virus was last propagated, it is not a heritable change but an epigenetic one.

A second system that bacteria possess to protect themselves from foreign DNA is the ability to detect 'foreign' modifications on such DNA and degrade it using specific endonucleases. For example, in the early days of recombinant DNA technology, it was difficult to isolate genes from animals and plants in the standard bacterial host, E. coli K-12. We now know that this failure was due to the destruction of the DNA because it contained methylated cytosine and was recognized by an endonuclease called Mcr (for methylcytosine restriction). This enzyme recognizes and cleaves any methylcytosine-containing DNA that does not have the E. coli K-12 signature cytosine methylation. Currently, E. coli strains lacking all known restriction systems are used to successfully isolate human and plant genes.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9780123739445000808

Electron Transport and Oxidative Phosphorylation

N.V. BHAGAVAN , in Medical Biochemistry (Fourth Edition), 2002

Transfer RNA (tRNA) Mutations

Fourteen mtDNA tRNA mutations have been associated with maternally inherited disease. Such mutations are typically associated with severe mitochondrial myopathies, characterized by "ragged red" skeletal muscle fibers upon Gomori trichrome staining and the accumulation of structurally abnormal mitochondria in muscle. Mutations in tRNAs exemplify the threshold effect whereby (due to replicative segragation) individuals may not exhibit clinical signs until the proportion of mutant mtDNA exceeds 80–90%. Myoclonic epilepsy and ragged red fiber (MERRF) disease, mitochondrial encephalomyopathy lactic acidosis (MELAS), as well as maternally inherited myopathy and cardiomyopathy (MMC) are well-characterized mitochondrial diseases.

Because oxidative phosphorylation capacity declines with age, individuals with identical mtDNA genotypes may express different clinical signs if they are of different ages. Individuals under the age of 20 with 80% mutant mtDNAs can be asymptomatic; individuals with the same mtDNA mutation over the age of 60 may suffer severe multisystem neurological disease. In general, older individuals have a lower threshold for oxidative phosphorylation dysfunction without clinical consequences.

Two tRNALeu(UUR) point mutations account for the majority of MELAS patients. An A-to-G transition at 3243 has been found in approximately 80% of MELAS patients. The 3243 mutation alters a highly conserved nucleotide within the dihydrouridine loop of the leucine tRNA. Unlike the MERRF variants, the 3243 mutation can result in a number of different clinical syndromes in addition to MELAS. These include diabetes mellitus and deafness, cardiomyopathy and ocular myopathy. Other mutations to tRNALeu result in mitochondrial myopathy as well as late-onset hypertrophic cardiomyopathy and myopathy. Overall, the mtDNA tRNA mutations affect both CNS and skeletal/cardiac muscle tissue while missense mutations primarily affect nervous tissue. Only one mitochondrial rRNA point mutation has been associated with disease. The homoplasmic A-to-G transition at position 1555 of the 12S rRNA gene is associated with maternally inherited deafness and aminoglycoside-induced deafness.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9780120954407500160

Scientific Fundamentals of Biotechnology

O.E. Tolmachov , in Comprehensive Biotechnology (Second Edition), 2011

1.06.16 DNA Cloning Using Homologous (General) Recombination

Conjugative DNA transfer and generalized transduction, the standard methods of classical bacterial genetics used to modify bacterial chromosomes, can be employed to engineer large bacterial plasmids. Essentially, large stretches of heterogeneous DNA are introduced into bacteria and recombine with the target plasmids via homologous recombination. The bacterial clones harboring the desired plasmid recombinants are selected using transferred genetic markers. Mutant bacteriophages P1vir and T4GT7 are common bacteriophages used for generalized transduction in E. coli.

Another typical approach for plasmid modification is the pop-in/pop-out technique, which can be used to insert any desired DNA sequence into a large plasmid. This strategy consists of the following three steps: (1) generation of the targeting construct, the plasmid, where the desired DNA fragment is flanked by the arms of homology to the recipient plasmid; (2) inter-molecular homologous recombination between the targeting construct and the recipient plasmid via one of the homology arms resulting in the co-integrate plasmid consisting of the recipient plasmid and the targeting construct (pop-in step); and (3) intra-molecular homologous recombination within the co-integrate plasmid via another homology arm resulting in the excision of the unwanted portion of the targeting construct with only the desired DNA fragment remaining inserted the original recipient plasmid (pop-out step).

Homologous recombination in wild-type E. coli is mediated by a number of cellular proteins with the central role played by the multifunctional RecA-recombinase. Enhanced homologous recombination can be achieved when the RecA protein is overexpressed from a multi-copy plasmid vector. A limitation of the RecA-mediated homologous recombination is the requirement for long (more than 500   bp) homology arms. An alternative recombinational pathway, mediated by the bacterial RecET-recombinase or closely related bacteriophage lambda Redαβ-recombinase, requires much shorter stretches of DNA homology (about 50   bp) making it a convenient choice for recombinogenic engineering (recombineering). In this scenario, which is called RecET-cloning, the targeting constructs can be routinely obtained by PCR amplification. The linear targeting construct is co-transformed with the linearized recipient plasmid and bacterial clones harboring the recircularized recombinant plasmids are selected using the marker of the recipient plasmid [42]. Linear DNA fragments are protected from degradation by exonucleases in bacteria using overexpression of the bacteriophage lambda gam product. It is not uncommon for such recombineering schemes to rely on several compatible plasmids coexisting in bacterial cells. Once the desired recombinant plasmid is generated, the bacteria can be cured of the unwanted plasmids either through their spontaneous loss or via high-temperature cultivation eliminating plasmids with temperature-sensitive origins of replication.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9780080885049000088

Conjugation

E.A. Raleigh , K.B. Low , in Brenner's Encyclopedia of Genetics (Second Edition), 2013

Formation of the Relaxosome

In preparation for DNA transfer (Dtr), a protein–DNA complex (relaxosome) is formed at a DNA region called oriT, which determines the site where one strand of the DNA is cut (nicked). This can be reconstructed in vitro in many cases, so the molecular stages of the process have been described in some detail ( Figure 2 ).

Figure 2. Relaxase formation for production of transfer strand during conjugation. Protein components are illustrated as follows: the bifunctional TraI phosphodiesterase (blue) with active site tyrosine (Tyr) and TraI helicase domains (light blue), IHF (orange), TraY (purple), TraM (green), and the T4CP TraD (yellow). At steps I and II, nickase in the context of other members of the relaxosome interacts with two distinct sites. At step III, the relaxosome is anchored at the membrane by interaction with T4CP. A signal presumed to indicate cell–cell contact (step IV) triggers rearrangement to begin translocation by the helicase domain of the nickase (step V); further rearrangement activates the ATPase activity of T4CP (TraD; step VI). Finally, an active complex of T4CP is formed (step VII), with recruitment of a second nickase to be transported into the recipient.

 Reproduced with permission from the American Society for Microbiology from Sut MV, Mihajlovic S, Lang S, Gruber CJ, and Zechner EL (2009) Journal of Bacteriology 191: 6888–6899.

In well-characterized examples, the nicking enzyme has two domains or components. One domain recognizes a part of oriT, while the other domain nicks nearby, becoming covalently attached to the 5′ end at the site of the nick. Additional proteins may be associated with this enzyme, forming a relaxosome that locally destabilizes the duplex and promotes the nicking activity. The assembled protein complex, or relaxase, may contain multiple subunits and may have regulated activity. Helicase activity is associated with the initiation of Dtr.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9780123749840003211

Agrobacterium and Plant Cell Transformation

P.J. Christie , in Encyclopedia of Microbiology (Third Edition), 2009

Definition of the T-DNA translocation pathway by TrIP

An assay termed transfer DNA immunoprecipitation (TrIP) was developed to trace the path of a DNA substrate through the T4S channel. TrIP, adapted from the chromatin immuoprecipitation assay, involves formaldehyde treatment of intact cells to cross-link channel subunits to the T-DNA substrate as it exits the cell, disruption of the cells, solubilization of membranes, and immunoprecipitation to recover channel subunits. The presence of T-DNA substrate in the immunoprecipitates is then detected by PCR amplification. With the TrIP assay, it was shown that the substrate forms close contacts with 6 of the 12 VirB/D4 components, VirD4, VirB11, polytopic VirB6, bitopic VirB8, VirB2 pilin, and VirB9. Analyses of various T4S mutants with the TrIP assay enabled formulation of a sequentially and spatially ordered translocation pathway for the T-DNA substrate. This pathway provides the first glimpse of how the T4S channel might be configured across the cell envelope ( Figure 5 ). The steps in the pathway are as follows:

•

Substrate recruitment. The T-DNA substrate binds VirD4 and it does so independently of other VirB proteins, establishing that VirD4 is the T-DNA receptor. A VirD4 Walker A mutant also retains T-DNA as well as protein substrate receptor activity, suggesting that binding of both types of substrates occurs independently of ATP energy.

•

Transfer to the VirB11 hexameric ATPase. Next, VirD4 transfers the T-DNA substrate to the VirB11 ATPase. This early transfer step also proceeds independently of ATP energy, as deduced by the finding that VirD4 or VirB11 Walker A mutations support substrate transfer. However, VirD4 cannot transfer the substrate to VirB11 in the absence of certain 'core' VirB proteins, suggesting that these core components are important for productive communication between VirD4 and VirB11.

•

Transfer to the integral IM proteins VirB6 and VirB8. VirB11 delivers the T-DNA substrate to the polytopic VirB6 and bitopic VirB8 proteins. VirB6 mutational studies identified a central periplasmic loop, termed P2, which is important for VirB6 binding to the DNA substrate. Other domains were implicated in regulating subsequent substrate translocation steps. Substrate transfer from VirB11 to VirB6 and VirB8 also require additional 'core' subunits, possibly important for VirB11 binding to these latter channel subunits, as well as the energetic contributions of VirB4, a third ATPase of this secretion system.

•

Transfer to the periplasmic and OM-associated proteins VirB2 and VirB9. VirB2 and VirB9 comprise the distal end of the T-DNA translocation pathway. As noted above, VirB2 polymerizes as the T pilus. Although it is formally possible that the T-DNA substrate moves through the lumen of the pilus to the plant cell, this probably is not the case because certain mutations block pilus production without affecting substrate translocation. In strains producing the 'uncoupling' mutant proteins, the cellular form of VirB2 is still required for substrate transfer. Thus, VirB2 might be a component of the secretion channel extending through the periplasm and, possibly, the OM. Several T4S subunits, including VirB3, VirB5, and VirB10, are required for this step of substrate transfer, but they do not form detectable interactions with the T-DNA. Therefore, VirB3, VirB5, and VirB10 are probably not channel subunits per se, but rather contribute to the structural integrity of the channel.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9780123739445001152

Vaccines and Clinical Immunization

Tak W. Mak , Mary E. Saunders , in The Immune Response, 2006

The efficiency of DNA transfer by bombardment varies with the device used and the type of target cell, but frequencies in the range of one stably transfected cell in 1,700–60,000 have been reported. These frequencies are in line with other methods of cell transfection such as electroporation or calcium phosphate precipitation. The viability of bombarded cells (80–95%) is also equivalent to that of cells transfected by other methods. However, gene gun methods use far less material than an in vivo vaccination procedure: while conventional injection methods require 10–100 μg of a vaccine plasmid to induce an immune response in a rat, gene guns can accomplish the same task using only 0.1–1 μg plasmid. Experiments using luciferase reporter gene systems have shown that transient expression of the gene of interest can first be detected 1–2 days after tissue bombardment into the skin, or into surgically exposed liver or muscle tissues of living rats or mice. DNA transfer occurs about 10 times more readily to skin and liver than to muscle; about 10–20% of cells in bombarded skin and liver express the transgene. Transient gene expression can be detected for at least 4–24 days after transfer, depending on the cell type targeted. Attempts to transfect human mammary gland explants and human endothelial, fibroblast, lymphocytic, and epithelial cell lines have also been successful. However, in the limited human studies available, it appears that higher amounts (500–2500 μg) than those used in rodents are required for in vivo human DNA vaccination resulting in measurable antibody and CTL responses. One last note: gene gun technology was first developed for the transfection of plant cells, and is used to great advantage in this field. Gene guns have also been used to introduce DNA into mitochondria and chloroplasts. [Plate courtesy of Dr. John O'Brien, www2.mrc-lmb.cam.ac.uk/personal/job.]

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9780120884513500259

Conjugation, Bacterial

L.S. Frost , in Encyclopedia of Microbiology (Third Edition), 2009

Transfer to Plants

The phenomenon of DNA transfer from A. tumefaciens to plant cells has features of both Gram-negative (pilus expression) and Gram-positive (induction of tra gene expression) bacteria and has been dealt with separately ( Figure 3 ). A. tumefaciens carrying large conjugative plasmids such as Ti (tumor-inducing) or Ri (root-inducing) greater than 200   kb cause crown gall disease in plants whereby they induce the formation of tumors at the site of infection. The Ti plasmid encodes a sensor-response regulator system, VirA and VirG, which in conjunction with ChvE, a chromosomally encoded periplasmic sugar-binding protein, process signals from wounded plant tissue. The phosphotransfer reaction from VirA to VirG induces gene expression from the virA, -B, -D, -E, and -G operons on the Ti plasmid. In addition, the virC, -F, and -H operons are induced but these operons express inessential gene products that affect host range or the degree of virulence. The signals generated by the plant include phenolic compounds, simple sugars, and decreased pH or phosphate content among others.

Figure 3. Signaling pathway used to stimulate T-DNA complex transfer to the plant nucleus and Ti conjugative transfer between Agrobacteria. Wounded plant tissue releases phenolics (acetosyringone) and sugars (1) that are detected by the two-component VirA and VirG regulatory system (2). This induces expression of the vir genes that encode the transfer apparatus at the pole of the cell that transports the T-DNA to the plant nucleus (3). The T-DNA is incorporated into the plant genome and produces the phytohormones auxin (indoleacetic acid) and cytokinin (4) that trigger tumorigenic growth of the plant tissue. The plant also produces opines (5) whose synthesis is encoded on the T-DNA. These unusual amino acids serve as a food source for Agrobacterium and also result in the induction of synthesis (6) of the conjugation factor, N-β-oxo-octanoyl-homoserine lactone (AAI; 7). AAI allows quorum sensing, which determines cell density with respect to Ti-plasmid-bearing cells resulting in conjugative transfer to other agrobacteria (8).

The virB region encodes 11 proteins that are homologous to the gene products in the Tra2 region of RP4 and are distantly related to the gene products of F. They encode the gene for prepropilin (VirB2), which is processed to pilin via a mechanism similar to that for RP4 pilin. A potential peptidase, homologous to TraF in RP4, has been identified (VirF) but its role has not been proven. The assembly of the VirB pilus is highly temperature dependent with an optimum of 19   °C, which is also maximal for the transfer process. The pili along with the T4SS transfer apparatus are localized to the pole of the cell, where transfer occurs.

The specific segment of single-stranded DNA that is transferred to the plant nucleus is called the T-DNA and can be characterized by the right (RB) and left (LB) borders, which are direct repeats of 25   bp. The T-DNA of nopaline-producing Ti plasmids is about 23   kb in length and contains genes for plant hormone expression (13   kb), a central region of unknown function, and a third region for opine (nopaline) biosynthesis (∼7   kb). The relaxase, VirD2, in conjunction with VirD1 that is similar to RP4 TraJ, cleaves at RB and subsequently LB in a TraI-like manner and remains attached to the 5′ end. VirC1, which binds to an 'overdrive' sequence near the RB, and VirC2 of certain Ti plasmids, enhance T-intermediate formation. Unlike other transfer systems, many copies of the T-DNA segment accumulate in the cytoplasm suggesting replacement replication is important in this system. The accumulation of T-DNA strands has been puzzling but might represent a strategy by the bacterium to ensure infection of the larger, more complex plant cell.

The DNA in the VirD2–T-DNA complex (T-complex) is thought to be coated with the single-stranded DNA-binding protein, VirE2, in preparation for transport through a conjugation pore composed of the VirB proteins (VirB2–VirB11). The VirD4 coupling protein (an ATPase) appears to work in conjunction with VirB11, another ATPase specific to P-like T4SS systems, to transport the T-complex ( Figure 2(b) ). Recent evidence suggests that VirE2 and VirD2–T-DNA transport are uncoupled and can occur using separate transfer pores. One of the Vir proteins, VirB1, which is inessential, resembles the transglycosylase of F (Orf169) and RP4 (TrbN) whereas a truncated version of VirB1 (VirB1*) is excreted into the rhizosphere and mediates adhesion between the bacterium at the site of transfer and the plant. Once the T-complex has entered the plant cytoplasm, the DNA is transported to the nucleus via nuclear localization signals (NLS) on the VirE2 and VirD2 proteins. The T-DNA is randomly integrated into the plant genome whereupon it begins to elicit signals for plant hormone production resulting in tumor formation. The T-DNA encodes for the synthesis of auxin (indoleacetic acid) and cytokinin isopentenyl adenosine, plant hormones that elicit uncontrolled growth at the site of infection. The bacteria derive nutrients from the tumor by the devious method of opine (unusual amino acid) production encoded by the T-DNA. Opines can be classified into about nine different types of compounds including octopine, nopaline, and agrocinopine, with up to three different opines being encoded by a particular T-DNA. Thus Ti plasmids are often referred to as octopine- or nopaline-type plasmids, for instance, depending on the opine they specify. The opines are excreted from the plant and taken up by the A. tumefaciens bacteria encoding a region on the Ti plasmid involved in opine utilization. The genes for opine catabolism (e.g., Occ for octopine catabolism) match the genes for the synthesis of that class of opines on the T-DNA.

An interesting aspect of Ti plasmid biology is the induction of conjugative transfer between agrobacterial cells in response to the presence of opines. The genes for this process (tra) are distinct from the genes for T-DNA transfer (vir) and encode a transfer region with homology to RP4 as well as the vir region itself.

Conjugative transfer by Ti has a narrow host range, limited to the genus of Agrobacterium. However, the host range can be extended to E. coli if an appropriate replicon is supplied, suggesting that it is plasmid maintenance and not conjugative functions that affect host range.

The process of inducing conjugative transfer in these bacteria is unique and fascinating. Initially, there is a low-level uptake of opines, which activates the regulatory protein OccR in octopine-type plasmids and inactivates the repressor protein AccR in nopaline-type plasmids such as pTiC58. This leads to increased expression of the tra and opine utilization genes by activation of TraR. TraI (not to be confused with relaxase proteins of F and RP4 plasmids) is a LuxI homologue that synthesizes a signaling compound, N-β-oxo-octanoyl-homoserine lactone (Agrobacterium autoinducer or AAI), belonging to a diverse class of homoserine lactone-like (HSL) compounds involved in quorum sensing or gene activation in response to changes in cell density ( Figure 3 ). TraR is a LuxR-like regulatory protein that detects increased levels of AAI and induces transfer gene expression to maximal levels. The result is the dissemination of the genes for opine utilization among the agrobacteria in the rhizosphere. Thus the system demonstrates a certain degree of chauvinistic behavior since the original colonizer of the plant cell shares its good fortune with its neighbors who then outcompete other bacteria in the rhizosphere.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9780123739445000079

Mitochondrial Medicine

Douglas C Wallace , ... Vincent Procaccio , in Emery and Rimoin's Principles and Practice of Medical Genetics (Sixth Edition), 2013

11.1.31 Diseases Resulting from Intragenic tRNA and rRNA Mutations

Intragenic mutations that alter mtDNA tRNA and rRNA genes and cause disease due to inhibition of mitochondrial protein synthesis are referred to as mtDNA protein synthesis defects. A defect in mitochondrial protein synthesis has the capacity to retard the synthesis of all of the mtDNA-encoded polypeptides, resulting in the inhibition of complexes I, III, IV, and V, but not II. However, since the largest and most numerous mtDNA-encoded subunits belong to complex I (7 subunits) and complex IV (3 subunits), intermediate-severity protein synthesis defects most commonly affect these enzymes.

Unlike mtDNA mutations that inactivate a particular polypeptide and result in more stereotypic clinical presentations, protein synthesis mutations are frequently associated with multisystem disorders. Their variable clinical phenotypes can be additionally complicated by variation in the mutant mtDNA heteroplasmy.

The most common clinical manifestation of mitochondrial protein synthesis mutations is MM involving RRF and abnormal mitochondria, frequently with paracrystalline precipitates. For some mtDNA protein synthesis mutations, this may be the primary clinical presentation. For protein synthesis mutations that result in multisystem encephalomyopathies at high percentages of mutant, patients with lower percentages of mutant mtDNA might only manifest MM.

The variability in clinical presentations of mtDNA protein synthesis mutations was already apparent in the first mtDNA protein synthesis mutation described, the MTTK∗MERRF8344G mutation, causing maternally inherited MERRF syndrome (5,6). Variability in clinical presentation is also the hallmark of the most common mtDNA protein synthesis mutation, the MTTL1∗MELAS3243G mutation, causing MELAS syndrome (326). At 10–30% mutation, the MTTL1∗MELAS3243G mutation causes diabetes mellitus with or without deafness. However, at higher percentages of mutant the same mutation can cause chronic progressive ophthalmoplegia, cardiomyopathy, or the complete MELAS syndrome (412).

In this section, we discuss the mitochondrial protein synthesis mutations classified based on the most common clinical presentation seen when the patients harbor a high percentage of the mutant mtDNA.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9780123838346000136

Scientific Fundamentals of Biotechnology

P. Chahal , ... A. Kamen , in Comprehensive Biotechnology (Second Edition), 2011

1.28.2.1.2 Electroporation

Electroporation was originally used to transfer DNA to Escherichia coli. Thereafter, it was adapted to eukaryotic cells as one of the efficient methods for delivering foreign genetic material to cells. The process forms pores in the cell membrane by the supply of an electrical pulse. Usually, cuvettes with a 0.4-cm electrode gap containing 0.5   ml of 2.0   ×   107 cell suspension/ml with about 30   µg of plasmid are shocked with an electric pulse generated by 260   V, infinite resistance, and 960   µF capacitance [5]. The duration and intensity of the electrical pulse are usually adjusted for a given cell type. This process is difficult to optimize and causes high mortality of cells at high-voltage exposure.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9780080885049000428

GENETIC AND CELLULAR THERAPIES

Ronald J Trent PhD, BSc(Med), MB BS (Sydney), DPhil (Oxon), FRACP, FRCPA Professor, Head , in Molecular Medicine (Third Edition), 2005

Strategies for Gene Delivery

There are two ways to transfer DNA (RNA) into cells— ex vivo or in vivo (Figure 6.3). A prerequisite for ex vivo transfer is the necessity to culture cells in vitro. Therefore, not all cells will be useful targets for this type of gene therapy. Another requirement is the ability to return the genetically altered cells to the patient; i.e., the cells need to be transplantable. The above considerations have meant that a lot of the work involving ex vivo transfer has focused on haematopoietic cells. Apart from the fact that ex vivo transfer may be the only suitable approach available in many cases, it has another advantage in terms of safety; i.e., there is more control over which cells will take up the foreign DNA. However, in vivo transfer is considered to be more physiological and may be the only option in some circumstances, e.g., disseminated cancer. In vivo transfer remains a priority awaiting further developments to ensure that the right cells express the transferred DNA, and they do so in adequate numbers. The concept of targeting becomes a real issue when in vivo transfer is considered (discussed further below).

Fig. 6.3. Transferring DNA into cells. Ex vivo : This approach involves the removal of cells from the patient. DNA (or RNA) is next introduced into the cells, which are then cultured to obtain adequate numbers. The introduction of DNA by infection using a viral vector is called transduction. The genetically altered cells (which may also be physically or antigenically altered following the ex vivo manoeuvres) are finally returned to the patient. In some circumstances, ex vivo transfer is the only feasible option, e.g., haematopoietic cells. In terms of safety, there is more confidence with ex vivo transfer since only the appropriate cells will take up the DNA/RNA. In vivo : A more physiological approach and challenge for the future is in vivo transfer, which involves direct entry of DNA (or RNA) into the patient. Targeting is necessary in this form of transfer.

The ultimate aim in gene transfer is to get DNA into specific tissues. Again there are two major approaches—physical and viral (biological) means. The cell and nuclear membranes can be made more permeable to DNA following co-precipitation of DNA with calcium phosphate, or an electric shock—called electroporation (Table 6.8). Using micropipettes, it is possible to inject DNA into the cell's nucleus. More novel approaches to facilitate movement of DNA into a cell include (1) injection of DNA directly into muscle cells; (2) insertion of DNA via cationic liposomes in the process known as lipofection, i.e., synthetic spherical vesicles that have lipid bilayers and so are able to cross the cell membrane and (3) coating of DNA with proteins and the "gene gun"— DNA-coated microprojectiles. Physical methods can be relatively inefficient when it comes to cells taking up DNA. More importantly, DNA inserted into the host genome in this way is usually present as multiple copies; i.e., there is no control over the sites of insertion, and so the function of normal genes could be affected. Finally, the expression of the introduced gene is only transient.

Table 6.8. Delivery systems for gene transfer A number of approaches can be used to get DNA into cells to allow genes to be expressed. The large number of options available would suggest that no single method is ideal. Even within the viral vectors, there are advantages and disadvantages found with each.

Type of approach Delivery method
Physical Greater membrane permeability to DNA: calcium phosphate coprecipitation, electroporation (electric shock)
Microinjection: into the cell nucleus, into muscles Other methods: insertion via liposomes, coating DNA with proteins, gene gun, microencapsulation
Viral Integrated into host's genome: retrovirus, adeno-associated virus, lentivirus
Not integrated into host's genome: adenovirus, herpes simplex 1 virus, a smallpox virus
a
The herpes simplex 1 based vector does not integrate (and so a transient effect would be expected), but the wild-type virus displays latency. Although the molecular basis for latency remains to be defined, it means that this virus could potentially be made to express its inserted gene over a longer time frame without the necessity for integration to occur.

The current preferred method for gene transfer involves the use of viruses, particularly the retroviruses. Wild-type retroviruses can convert their RNA into double-stranded DNA, which can then integrate into the host's genome (see Figure 8.2). Viral proteins encoded by the gag, pol and env genes make up approximately 80% of the retroviral genome. These RNA segments can be deleted and replaced by a foreign gene, e.g., human adenosine deaminase (ADA). Now the recombinant retrovirus is no longer infectious because it cannot make its own structural proteins. This is a prerequisite for gene therapy. Persistent infection by the genetically engineered retrovirus would not be permissible since it might lead to neoplastic change, the wrong cells expressing the gene, or the germ cells becoming infected and so passing on the gene or any genetic defects created to future generations. To become a useful vector for DNA transfer, the retrovirus must infect in a controlled way. This can be done with packaging cells. These cells contain a helper retrovirus that has also been genetically manipulated to produce empty virions; i.e., structural proteins are present, but a complete infectious virion cannot be made. However, the retroviral vector with its inserted ADA gene can utilise the structural proteins produced by the helper virus in the packaging cells to form a complete (infectious) virion, which can undergo one round of infection. This would be enough to get the genetically engineered retroviral RNA into the target cells' DNA. Advantages of the retroviral vector for DNA transfer include (1) A single virus infects one cell. (2) The virus is usually non-immunogenic. (3) Integration into the host genome means there is the potential for long-term expression of the inserted gene. Disadvantages are (1) The target cell must be dividing before the retrovirus can integrate into the cell's genome. (2) Transduction efficiency is usually inadequate. (3) DNA insert size is limited, which can be a problem if a large gene is involved. (4) Since retroviral vectors are produced from living cells, there is the worry that contaminants derived from these cells will be present.

Risks are involved with using retroviruses as gene transfer vectors: (1) Integration is random, and so there is always the worry that a normal gene is inactivated or an oncogene is activated. (2) Retroviruses have the potential to revert to replication-competent organisms and so induce cancer (Figure 4.8). Because of these issues, a number of other viruses have been developed for gene therapy. Two of them (adeno-associated virus and lentivirus) will integrate into host DNA and so can lead to long-term expression of the transduced gene; i.e., a cure is possible. The latter goal must be balanced by long-term side effects if the integration has interfered with the function of a normal gene. The other viral vectors (as well as the physical means) do not lead to integration, and so the associated genes are expressed for only a short term; i.e., treatment will need to be repeated (Table 6.9).

Table 6.9. A comparison of different methods for introducing DNA in gene therapy

Method a Target cell Chromosomal integration (gene insert size in kb) Other advantages Other disadvantages
Retrovirus Limited to dividing cells Yes (medium-sized insert ~8 kb) Easy to manipulate. Considerable experience because used in many trials. Potential for long-term gene expression. Known to cause disease in humans, i.e., risk if recombination occurs as non-random integration means endogenous genes can be disrupted.
Adenovirus Both dividing and non-dividing cells No, episomal (medium-sized insert ~8 kb) Easy to manipulate. Can achieve high titres in production. Used in many trials. Does not integrate, i.e., no risk of insertional mutagenesis Potentially immunogenic and can provoke toxic response in host (current batches of this virus less immunogenic). Short-term expression.
Adeno-associated virus Both dividing and non-dividing cells Yes (small insert size ~5 kb) Not known to cause disease in humans. Non-immunogenic. Relatively new. Difficult to purify. Lack of expressed viral protein means limited immune responses provoked. Potential for insertional mutagenesis. Detected in semen but unclear if in sperm or tissues or fluids.
Lentivirus Both dividing and non-dividing cells Yes (medium-sized insert ~8 kb) Non-immunogenic. Easily manipulated. Long-term gene expression. Derived from HIV and so concern that wild-type recombinant is formed. Potential for insertional mutagenesis. Difficult to manufacture.
Herpes simplex virus Both dividing and non-dividing cells Extra-chromosomal (large insert size >30 kb) Does not disrupt endogenous genes, i.e., remains extrachromosomal. Potential for latency, i.e., long-term expression. Limited use if patient already has developed immunity to this virus.
Naked plasmid DNA Most cells No (relatively large inserts >10 kb) Can be inserted into many cells by various means. Easy to make. Minimal biosafety risks. Larger than oligonucleotides and so requires some packaging. May provoke immune response. Generally DNA vaccines are safe but ineffective.
a
The transmission of genetic material from one cell to another by viral infection is called transduction. Acquisition of new genetic markers by incorporation of added DNA into eukaryotic cells by physical or viral means is called transfection.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9780126990577500060