Vectors gene insertion-DNA Cloning with Plasmid Vectors - Molecular Cell Biology - NCBI Bookshelf

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Vectors gene insertion

Vectors gene insertion

Vectors gene insertion

Vectors gene insertion

Privately sent vibrator completely synthetic dsDNAs can be cloned into plasmid vectors just as DNA restriction fragments prepared from living organisms are. Similarly, to overcome the risk for activation of neighboring genes following vector integration, self-inactivating vectors are being gdne to have diminished ability to activate genes over long distances [], although Vectors gene insertion is not clear whether these vectors will be safer [ ]. Gene therapy vectors are extensively minimized to eliminate their replicative potential and reduce their collateral effects on the target onsertion [ 15 ]. Analysis of the bendability of all SB sites mapped in the screen reported by Yant and coworkers Eros rammazoti a peak at the center of the insertion site that is defined by the central TA dinucleotide. Viral and transposon vectors have been employed in gene therapy as well as functional genomics studies. Although a Vectors gene insertion for transcriptional units might seem beneficial for functional genomics studies, the myriad of recently identified noncoding Insertuon genes [ ] as well as other RNA product genes such as those encoding rRNA and tRNAs involved in gene regulation may not be targeted by viral vectors that preferentially integrate into or near protein encoding genes. Delivery of nonviral DNA into mammalian genomes involves avoiding or traversing numerous barriers, including enzymes in the blood and cellular environments, the endothelial lining of vessel walls, cellular plasma membranes, Vectrs membranes, nuclear membranes, and chromosomal integrity [ 71 ]. This genr is done by another enzyme carried in the virus called integrase see figure 2. Figure General procedure for cloning a DNA fragment in a plasmid vector.

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Avery, C. In addition, great care should be taken to provide balanced views of the risks associated with the gene-transfer techniques and to abstain from sweeping generalizations. The four major types of vectors are plasmidsviral vectorscosmidsand artificial chromosomes. Because the virus has been adapted to lose most of its genome, the virus becomes safer and more effective in transplanting the required genes into the host cell. It seems, therefore, justified to assume that the use of other vector systems that introduce naked DNA eg plasmids R tutorial linear model nuclei will also be accompanied by the integration of vector sequences into the host-cell genome. If one assumes the integration frequencies in vivo to be equivalent to those observed in cell lines, then the vector amounts used for in vivo gene therapy up to 10 13 vector-particles per dose would imply that a significant number of cells even millions may acquire genome-integrated vector fragments. All non-recombinant phage go lysogenic and produce no viral progeny. Transposon Tc1 of the nematode Caenorhabditis elegans Vectors gene insertion in human cells Nucleic Acids Res 26 : — As murine retroviruses need both nuclear membrane degradation and active DNA synthesis in order to integrate, 1 their use is limited to applications in which mitotically active cells are to be modified. Because the DNA isolated from an individual organism has a specific sequence, restriction enzymes cut Vectors gene insertion DNA into a reproducible set of fragments called restriction fragments Figure Yang J et al. In the case of DNAthis is feasible for relatively short molecules such as the genomes of small viruses.

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  • Specifically, a cloning vector is DNA taken from a virus, plasmid, or cells of higher organisms to be inserted with a foreign DNA fragment for cloning purposes.
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Help us improve our products. Sign up to take part. It goes without saying that efficient gene transfer is essential to gene therapy.

Many different gene-transfer systems are being developed for numerous applications. Vector systems are usually divided into those where the vector inserts itself into the host genome and those with vectors that remain extra-chromosomal. Integrated vectors are the system of choice if persistent expression is required. However, their insertion results in an alteration of the chromosomal DNA and therefore may lead to insertional mutagenesis.

Although the risk of insertional mutagenesis is a recognized disadvantage, many gene therapy protocols embrace the use of integrating vectors. Apparently, the small possibility of adverse effects of insertion mutagenesis is not seen as a major obstacle for the use of integrating vectors for clinical gene therapy. Retroviruses are the archetype for vectors that integrate themselves into the host-cell genome.

The murine retroviruses from which they are derived are notorious for their propensity to induce tumors in their natural hosts hence their classification in the subfamily Oncovirinae.

Once integrated, the provirus can perturb the expression of nearby cellular genes. In some instances, such a mutation can constitute a discrete step in the multistep process that eventually leads to cellular transformation. The unequalled efficiency with which retroviral vectors integrate their genome into the host-cell chromosomal DNA has made them the system of choice for many gene therapy applications.

They have been used extensively in human gene therapy research. In more than clinical trials, over patients have received retrovirus vectors. So far, not a single case has been reported in which adverse events have been attributed to insertional mutagenesis.

As murine retroviruses need both nuclear membrane degradation and active DNA synthesis in order to integrate, 1 their use is limited to applications in which mitotically active cells are to be modified. The relatively short half-life of the pre-integration complex limits the window during which infection leads to provirus integration to only a few hours.

In contrast to the murine retrovirus-derived vectors, lentivirus vectors are suitable for gene transfer into nonmitotic, quiescent cells, as their pre-integration complex can cross the nuclear membrane.

Therefore, lentivirus vectors appear more suitable for in vivo gene transfer and major research efforts have been instigated to develop safe and effective vector systems. The adeno-associated virus AAV has also been used as a vector.

AAV is a member of the Dependovirus group of the parvovirus family. This virus requires the presence of adenovirus or herpesvirus as helper, for productive infection. In the absence of helper virus, the viral genome can integrate into the cellular DNA.

In the presence of the AAV-derived rep protein, the viral genome integrates preferentially at one locus on human chromosome Upon subsequent infection with the herpes or adenovirus helpers, AAV can be activated. Although evidence suggests that at least a fraction of the AAV vectors persist as integrated copies, significant amounts of vector DNA appear to be maintained as large episomal concatemers. At present, the above-mentioned integrating vectors are the most widely used, but there are also ongoing efforts to generate hybrid vectors, which would be able to integrate into the genome of nonmitotic cells.

Several attempts have been made to provide nonintegrating vectors eg adenovirus vectors , which have the capacity to transduce nonmitotic cells, with integration mechanisms in order to insert the transgene into the host-cell genome. This would achieve the best of both worlds. The adenovirus—retrovirus chimeric vectors are an example of this new approach. This system consists of adenovirus vectors, which carry the gag , pol and env genes required for packaging the retroviral vector genomes, and an adenovirus vector that contains a recombinant provirus.

When cells are co-infected with the above vectors, the transduced cells start to shed progeny retroviral vectors which can infect and stably modify the neighboring cells. In this way, the high titer of adenoviruses is combined with the integration capacity of retroviruses.

Note that with this strategy only mitotically active cells can be stably modified. A strategy that should be applicable to nonmitotic cells too, has recently been reported and involves the use of an AAV—adenovirus hybrid. However, in the latter study no evidence was presented confirming the integration of the hybrid vector-derived AAV cassette.

Similarly, integration or recombination systems of other origins have been exploited for integration of transgenes into the host cell genome. Consequently, such hybrid vectors have the risk of insertional mutagenesis in common with the more conventional integrating vectors. The absence of integration is often used as a safety argument to promote the use of non-integrating vectors, such as adenovirus-derived vectors or plasmid vectors. This argument should be used with caution.

Adenovirus DNA can recombine with chromosomal DNA, and as a result, vector sequences can become integrated into the host-cell genome. However, these frequencies have been determined in established cell lines, and it is not clear whether it is justified to extrapolate these frequencies to the in vivo situation and to estimate the risk.

It would be very interesting to know the frequency with which the adenovirus vectors integrate into the host-cell genome of diploid cells. In a study with E1-containing, but replication-defective adenovirus vectors in cultures of rat diploid kidney cells, Fallaux et al 10 reported the frequent occurrence of transformed foci.

This indicates that in diploid cells too, the E1 region can become integrated into the host-cell genome as a result of illegitimate recombination, although the E1 proteins might influence the frequency of integration. In rodents and in hamsters, adenoviruses can also become integrated into the host-cell genome, as is evidenced by the occurrence of E1- containing tumors upon injection of, for example, subgroup-A adenoviruses. If one assumes the integration frequencies in vivo to be equivalent to those observed in cell lines, then the vector amounts used for in vivo gene therapy up to 10 13 vector-particles per dose would imply that a significant number of cells even millions may acquire genome-integrated vector fragments.

Thus, it is not strictly accurate to categorize adenovirus vectors as being non-integrating, when considering the risk of insertional mutagenesis. It should be noted that the insertion of vector sequences is not provoked by the adenovirus but, rather, is the result of illegitimate recombination.

It seems, therefore, justified to assume that the use of other vector systems that introduce naked DNA eg plasmids into nuclei will also be accompanied by the integration of vector sequences into the host-cell genome. However annoying this may seem at first sight, it should be seen in its proper perspective. In healthy human cells, chromosomal DNA is definitely not static and subject to mutation and recombination.

Also germ line and somatic cells are modified, at a fairly high frequency, by transposable elements. As long as the potential benefit outweighs the risk one should not hesitate to choose gene therapy for the treatment of severe disorders, but it is essential to ensure a proper follow-up of all patients who participate in clinical trials.

In addition, great care should be taken to provide balanced views of the risks associated with the gene-transfer techniques and to abstain from sweeping generalizations.

If given proper counseling, the people involved can make their judgments based on their personal perception of the risk and benefits. We should realize that we are making such judgments every day.

Don't most of us expose ourselves to sunlight to acquire a tan, at the cost of an increased risk of skin cancer due to UV-induced mutagenesis?

Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection Mol Cell Biol 10 : — Lentiviral vectors — the promise of gene therapy within reach? Science : — Yang J et al. Concatamerization of adeno-associated virus circular genomes occurs through intermolecular recombination J Virol 73 : — Recchia A et al. Fraefel C et al. Zhang L et al. The Himar1 mariner transposase cloned in a recombinant adenovirus vector is functional in mammalian cells Nucleic Acids Res 26 : — Schouten GJ et al.

Transposon Tc1 of the nematode Caenorhabditis elegans jumps in human cells Nucleic Acids Res 26 : — Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells Cell 91 : — Frequency and stability of chromosomal integration of adenovirus vectors J Virol 73 : — Who's afraid of replication-competent adenoviruses? Gene Therapy 6 : — Kazazian HH Jr.

An estimated frequency of endogenous insertional mutations in humans Nat Genet 22 : Jonason AS et al. Download references. Reprints and Permissions. Human Gene Therapy The American Journal of Pathology Molecular Therapy - Nucleic Acids Nature Protocols Advanced search.

Skip to main content. Insertion vectors for gene therapy It goes without saying that efficient gene transfer is essential to gene therapy. Rights and permissions Reprints and Permissions. Download PDF. Gene Therapy menu. Nature Research menu. Search Article search Search.

WordPress Shortcode. Molecular Cell Biology. Lentivirus are a family of viruses that are responsible for notable diseases like HIV , which infect by inserting DNA into their host cells' genome. Uptake of plasmids by E. To understand the capabilities of a lentiviral vector , one has to consider the biology of the infection process. This argument should be used with caution.

Vectors gene insertion

Vectors gene insertion

Vectors gene insertion

Vectors gene insertion

Vectors gene insertion

Vectors gene insertion. Insertion vectors for gene therapy

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Gene Therapy Viral Vectors Explained

Metrics details. Viral and transposon vectors have been employed in gene therapy as well as functional genomics studies. However, the goals of gene therapy and functional genomics are entirely different; gene therapists hope to avoid altering endogenous gene expression especially the activation of oncogenes , whereas geneticists do want to alter expression of chromosomal genes.

The odds of either outcome depend on a vector's preference to integrate into genes or control regions, and these preferences vary between vectors. Here we discuss the relative strengths of DNA vectors over viral vectors, and review methods to overcome barriers to delivery inherent to DNA vectors.

We also review the tendencies of several classes of retroviral and transposon vectors to target DNA sequences, genes, and genetic elements with respect to the balance between insertion preferences and oncogenic selection. Theoretically, knowing the variables that affect integration for various vectors will allow researchers to choose the vector with the most utility for their specific purposes. The three principle benefits from elucidating factors that affect preferences in integration are as follows: in gene therapy, it allows assessment of the overall risks for activating an oncogene or inactivating a tumor suppressor gene that could lead to severe adverse effects years after treatment; in genomic studies, it allows one to discern random from selected integration events; and in gene therapy as well as functional genomics, it facilitates design of vectors that are better targeted to specific sequences, which would be a significant advance in the art of transgenesis.

Elements such as viruses and transposons, through evolution with their host organisms, have acquired the ability to integrate into host genomes and ultimately shuffle genetic material between organisms. These elements have an established history in molecular biology and genetics research because of their ability to deliver specific genetic cargo, randomly disrupt host genomes for genetic screens, and serve as vectors for delivery of therapeutic expression cassettes to treat human disease.

Viral vectors have been the predominant tools for these applications for three reasons: the ease and efficiency with which specific viral genetic cassettes can be introduced into cells; the vast accumulated knowledge of viruses and their mechanisms of gene transfer into chromosomes; and the large number of sites in genomes into which they can integrate. Retroviruses in particular have been used for random insertion into chromatin to interrupt host genes insertional mutagenesis and thereby identify their function [ 1 — 3 ] as well as for delivery of therapeutic genes [ 4 — 6 ].

Moreover, viral activation of oncogenes and, more recently, inactivation of tumor suppressors have been used to discover several novel genes that are involved in cancer progression [ 7 — 12 ]. The potential genetic consequences of insertions of integrating vectors are summarized in Figure 1. Potential genetic consequences of integration of transgenic cassettes into chromatin.

An expression cassette orange box in a viral or nonviral vector represented by purple inverted arrowheads, which indicate either inverted or direct terminal repeats can integrate into four classes of chromatin. Regulatory elements in the transgenic cassette may either enhance expression of the neighboring gene GOF for gene X or, in rare cases, block expression of an active gene. Integration within some genes can also lead to a dominant gain of function DGF or production of a dominant-negative form DNF of the original gene X.

A further discussion of effects of insertional mutagenesis can be found in the reports by Carlson and Largaespada [61] and Collier and Largaespada []. Activation of oncogenes in mice by insertionally mutagenic retroviruses suggested that inadvertent oncogene activation resulting from the use of relatively benign therapeutic vectors is a potential risk associated with gene therapy. Gene therapy vectors are extensively minimized to eliminate their replicative potential and reduce their collateral effects on the target genome [ 15 ].

However, extensive testing in animals demonstrated that the risk of oncogenic activation was real, although variable and dependent on the viral vector used, the genetic cargo, and the background genetics of the model system [ 16 — 22 ]. Given what was assumed to be acceptable risk, retroviral gene therapy trials have been conducted in human patients. Nearly 1, clinical gene therapy trials have been initiated, more than half with retroviral vectors [ 4 ], but as yet no vectors have been approved in the USA for clinical gene therapy outside the clinical trial setting [ 23 ].

Gendicine, an adenovirus designed to restore p53 function in cancerous cells, has been approved for commercial human gene therapy in China [ 24 ], although this vector is essentially nonintegrating and thus carries decreased risk for oncogene activation via vector insertion. The worst fears of the gene therapy field, oncogene activation, were realized when three of more than 20 patients treated for X-linked severe combined immunodeficiency disease X-SCID developed leukemia.

These adverse findings, including one death, occurred 3 years or more after administration of therapeutic murine leukemia virus MLV -derived retrovirus vectors [ 25 , 26 ]. The linkage between treatment and leukemias could be inferred because the expanded transformed cell populations harbored clonal integrations of the therapeutic vector, which suggested a biologic selection for the retrovirus-induced mutation [ 27 — 30 ]. However, these studies also indicated that clonal expansions in some cases appeared to be temporary and did not always lead to adverse effects, features that could actually improve the likelihood of successful gene therapy.

The cause of at least two of the leukemias appears to be insertion of the MLV vector close to the LMO2 oncogene, which led to LMO2's activation by enhancers in the long terminal repeat LTR sequences of the vector [ 31 — 33 ]. Retrospective examination of the role in LMO2 during development supported this conclusion [ 34 , 35 ]. The relevance of these observations to clinical cases, however, is highly debatable [ 37 , 38 ].

In contrast, other gene therapy trials that employed retroviral vectors to treat adenosine deaminase deficiency [ 39 — 41 ] and chronic granulomatosis disease CGD [ 42 ] have not yet reported any equivalent adverse events. As noted previously, findings of preferential integration around certain genes is not necessarily due to a preference for these genes, but may rather be a consequence of clonal expansion that can be transient and thereby beneficial in terms of enhancing the number of therapeutic cells.

A similar effect has also been observed in nonhuman primate studies, indicating that this result may not be unique [ 19 ]. Despite the striking incidence of common integration sites that are often associated with tumor or leukemia formation [ 8 , 47 , 48 ], there has been no report of adverse events in the CGD patients and no indication that the corrective gene, gp91 phox , synergizes with any of the three common integration site genes to promote growth.

Taken together, the results of the CGD and X-linked plus adenosine deaminase SCID trials demonstrate that oncogenesis is not necessarily an inherent, inevitable side effect of gene therapy.

However, tumors and leukemias can take years to manifest, and these trials are in their early years. A clearer understanding of the variables that underlie oncogenesis is needed in order to increase the safety of these trials. These variables include insertion site preferences of therapeutic vectors, their abilities to activate nearby genes, and interactions between specific genetic cargos and activated host genes.

Although cargo-host interactions will be specific to each gene therapy approach, the vectors themselves govern other parameters of insertion preference and neighboring gene activation. Analyses of insertion preferences, in particular, have received much recent attention, and have sparked interest in the use of transposons as alternatives to viruses as gene therapy vectors. Transposable elements also have been used for insertional mutagenesis and genetic studies in model organisms, and are being developed as gene therapy agents in humans [ 50 — 53 ].

The most well characterized DNA transposon vector used in mammals is the synthetic Sleeping Beauty SB transposon system [ 54 ], which over the past decade has become a powerful tool in functional genomics to identify genes in vertebrates, including fish and mammals [ 55 — 61 ]. Application of transposon-mediated gene transfer to gene therapy has been explored because it avoids several disadvantages of viral delivery systems. These disadvantages of viruses include the following: 1 their preference for integrating into genes [ 62 — 65 ]; 2 the difficulty with purification to eliminate toxic or infectious agents [ 66 ]; 3 their potential to elicit unwanted immune or inflammatory responses [ 67 , 68 ]; 4 the constraint on therapeutic cargo size; and 5 the difficulty and expense associated with their production in large quantities [ 69 , 70 ].

In contrast to viral vectors, preparations of nonviral plasmid-based transposon vectors are relatively inexpensive to purify, are largely nonimmunogenic, and have no hard constraints on genetic sequences that can be delivered. A negative tradeoff with DNA vectors is increased difficulty in delivery.

Delivery of nonviral DNA into mammalian genomes involves avoiding or traversing numerous barriers, including enzymes in the blood and cellular environments, the endothelial lining of vessel walls, cellular plasma membranes, endosomal membranes, nuclear membranes, and chromosomal integrity [ 71 ]. There are three delivery approaches that work across the nanoscale, microscale, and macroscale [ 72 ]. The nanoscale approach comprises delivery of single or small numbers of DNA molecules, which most often are collapsed by polycationic polymers for example, polylysine and other modified amino acids, and various linear and branched forms of polyethylenimine, among others or lipids, with or without various ligands for review, see the report by Wagner and coworkers [ 71 ].

Some polycationic complexes are cytotoxic or unstable in the blood, which can be circumvented by encasing the complexes in polyethylene glycol [ 73 ]. In mice, the most effective method for in vivo gene transfer and expression has been demonstrated in hepatocytes using simple infusion of naked plasmid DNA under increased pressure.

Achieving a clinically feasible method of local delivery to liver in large animals, including humans, is a challenge that is being addressed by more localized hydrodynamic delivery using specialized catheters or pressure cuffs [ 77 , 78 ]. On the microscale, condensing DNA with polyamines such as polyethylenimine to a complex small enough to be taken up by cells into endosomes has been studied intensively [ 79 , 80 ].

Our findings Hackett PB, Podetz-Pedersen K, Bell JB, McIvor RS, unpublished data suggest that gene expression following hydrodynamic delivery is about fold more effective than delivery using polyethylenimine [ 81 , 82 ] and only about fold to fold less effective than viral delivery to liver [ 72 ].

Alternative delivery ex vivo using electroporation is under development and has been achieved in hematopoietic stem cells [ 83 ]. Since the development of the SB system, nonviral, integrating DNAs have established themselves as potential vectors for gene therapy.

Following hydrodynamic delivery, transposons have been used in mice to cure hemophilias A and B [ 84 — 87 ] and tyrosinemia type I [ 88 , 89 ]. Other somatic delivery methods were used to ameliorate blistering skin disease junctional epidermolysis bullosa [ 90 ], retard glioma xenographs [ 91 , 92 ], produce Huntingtin protein in a model of Huntington disease [ 93 ], and as a preventive treatment for lung allograft fibrosis [ 94 ].

Based on the findings summarized above, we estimate that only about one in 10, SB transposons that are delivered to liver or lung actually transpose into chromatin Hackett PB, unpublished data. This procedure is sufficient to cure diseases such as hemophilia and tyrosinemia type 1, and to ameliorate other diseases such as mucopolysaccharidoses types I and VII. Although quantifying the number of transposon insertions per cell has not been done because of the difficulty of cloning insertion sites in mostly nondividing cells in most organs of animals, the expression data are consistent with a single integration in most if not all transgene-expressing cells.

In addition to SB, several other transposon vectors and phage integrase-based vectors have been tested for their potential to deliver therapeutic genes, including Frog Prince [ 96 ], Tol2 [ 89 ], and piggyBac [ 97 ], as well as other well characterized transposons such as the Drosophila P-elements, which are not mobilized very efficiently in mammalian cells [ 98 ].

These vectors differ in their efficiency of gene insertion, genetic cargo capacity, integration site preferences, and effects on chromosomal stability.

Among other advantages these systems have over retroviruses as gene therapy vectors, transposons present a wide variety of insertion site preferences that differ from those of retroviruses, with possible consequences for oncogene activation. The characteristics of these vectors are summarized in Table 1.

The remainder of this review discusses these differences as they relate to gene therapy and functional genomics. Although most vectors will integrate into a vast number of sites scattered throughout the genome, numerous studies have shown that these integrations are not random with respect to several variables. Global preferences for vector integration can be governed by large-scale genomic context such as coding and regulatory regions of genes, and their transcriptional status, as compared with intragenic regions [ 99 ].

The fine tuning that determines specific sites of integration is governed by smaller scale, physical features, such as the specific sequences of nucleotides surrounding insertion sites and DNA structural characteristics derived from these sequences.

Figure 2 illustrates some of the physical features of DNA that are influenced by local sequence. The figure illustrates physical parameters of B-form DNA structure that are altered in preferred sites for integration of insertional vectors. A given base pair may be distorted in more than one of these parameters. V step analysis is a method of examining these, and other physical parameters such as 'shift', in terms of a single number that derives from the transition from one base pair to another [,].

Viruses and transposons exhibit a wide range of variability with respect to preference for genes and transcriptional units. Several studies have mapped hundreds to thousands of insertions into human or mouse genomes, and correlated insertion positions with known genes.

Many retroviruses exhibit a nonrandom preference for genes [ 65 ]. This could be due to greater accessibility of the DNA in 'open' chromatin or interaction of integrase enzymes with cellular factors bound to transcriptional regulatory elements. In a similar approach using the SB transposon, Yant and coworkers [ ] found that SB exhibited a much lower although nonrandom preference for genes.

Although a preference for transcriptional units might seem beneficial for functional genomics studies, the myriad of recently identified noncoding RNA genes [ ] as well as other RNA product genes such as those encoding rRNA and tRNAs involved in gene regulation may not be targeted by viral vectors that preferentially integrate into or near protein encoding genes.

Targeting of various vectors to these non-coding RNAs in gene therapy, and any resulting deleterious effects, has not been extensively examined.

Many vectors appear to exhibit a preference for specific genes. In insertional mutagenesis studies, the identification of recurrent viral insertions into a specific group of genes was taken to mean that viral activation of these putative oncogenes in individual cells led to clonal expansion among a pool of cells in which every host gene was an equal target for integration as discussed above for LMO2.

However, when MLV insertions were mapped in normal HeLa cells that did not undergo any type of selection, oncogenic or otherwise, many of these same genes harbored recurrent integrations, suggesting that vectors may inherently target specific genes [ 48 ].

The basis of this selection is not understood, but it may be similar to that discussed above for HIV. In addition to general preferences for genes, many viral vectors, including retroviruses, lentiviruses, and adeno-associated virus, preferentially target transcriptional units or their promoters.

MLV retroviruses have a preference for integration proximal to transcriptional initiation sites [ 64 , 65 , — ], which is a problematic trait, considering that MLV-based vectors are the most commonly used vectors in human gene therapy [ 4 ]. HIV and adeno-associated viruses have preferences for entire transcriptional units [ , , — ] see Note added in proof, below ; this is in contrast to MLV, which targets only the region proximal to promoters.

Additionally, expression array studies have shown that HIV has a preference for transcriptionally active genes [ 65 ] as well as an avoidance of chromatin regions in which transcription is repressed [ ]. In contrast to these viral vectors, SB transposons and avian leukosis virus a retrovirus apparently have only a slight preference for either transcriptional units or their regulatory elements [ , ], with little or no preference for transcriptionally active genes [ 65 ].

In one survey, SB exhibited an overall preference for microsatellite repeats, found primarily in noncoding regions [ ], possibly due to the preferred target sites found in TA repeats [ ].

A study that correlated insertions sites with hundreds of genome annotations [ 99 ] illustrated the degree to which genomic features and primary sequence influenced vector integration preferences for several vectors for example, the L1 and SB transposon insertions were much more influenced by primary sequence than were retroviral vectors.

This study also found variable preferences between vectors for elements such as CpG islands, DNase I sensitive sites, and transcription factor binding sites. The recent identification of a periodic sequence encoding nucleosome positioning [ ] may also correlate with vector integration patterns, because nucleosomes have been shown to affect patterns of retroviral integration [ ].

Similar studies to identify trends for piggyBac and Tol2 with respect to genome-wide integration preferences will be valuable in assessing the relative safety of these vectors for gene therapy.

Although many vectors exhibit a preference for genes, and even specific genes, few vectors repeatedly integrate into the same precise position with any significant frequency. Rather, most genes harboring frequent insertions show a distribution of insertions into several positions within the same gene. Because the oncogenic potential of a vector is related to its propensity to integrate in or near a select few genes, understanding local parameters that affect integration may contribute to our ability to assess the risk associated with these vectors in gene therapy.

Vectors gene insertion

Vectors gene insertion