gene transfer technology requires the introduction of recombinant genetic elements into human cells and holds a considerable therapeutic potential for the treatment of a wide variety of pathological conditions , such as cancer , genetic disorders , neurological illnesses , diabetes , infectious diseases , and cardiovascular maladies [ 17 ] . the gene - based treatments both of cancer and infectious diseases may require only a transient expression of the recombinant genetic elements , which have the function to destroy either neoplastic tissues , or cells that harbor an infectious agent [ 3 , 4 ] . conversely , genetic disorders , neurological illnesses and cardiovascular maladies need a long - term transgene expression , as the treatment of these maladies envisions the introduction of functional copies of certain genes in the attempt to correct the phenotype of the disease [ 3 , 4 ] . a long - term transgene expression is also required for autologous t lymphocytes that are genetically engineered to express recombinant t cell receptors , which may impart binding specificity either for neoplastic markers or infected cells [ 57 ] . to this end , gene delivery systems that integrate their genome into the target cell chromosomal dna allow for a more stable and long - lasting transgene expression [ 3 , 4 ] . various types of gene delivery systems are currently available [ 3 , 4 , 816 ] . the most common gene transfer models derive from retroviruses , lentiviruses , adenoviruses , adeno - associated viruses ( aav ) and so - called non - viral vector systems [ 3 , 4 , 816 ] . adenoviruses and most of nonviral - derived vectors are episomal gene transfer models [ 3 , 4 , 13 , 14 ] , whereas vectors based on retroviruses , lentiviruses , aav , sleeping beauty ( sb ) dna transposon system , and steptomyces bacteriophage integrase c31 have integrative properties [ 3 , 4 , 12 , 1416 ] . sleeping beauty ( sb ) dna transposon system and streptomyces bacteriophage integrase c31 are non - viral - derived gene transfer systems that only require a plasmid dna transfection for the delivery of their transgene , which is then stably integrated within the target cell genome . retroviral - based vector systems were utilized in the first two phase i gene therapy clinical trials , which were conducted in the united states of america between 1989 and 1990 [ 3 , 4 , 810 ] . one clinical trial dealt with the treatment of adenosine deaminase ( ada ) deficiency , which is a genetic disorder leading to immunodeficiency [ 3 , 4 , 9 , 10 ] . a retroviral vector carrying the functional copy of adenosine deaminase was used for the ex vivo gene transduction of autologous bone marrow - derived hematopoietic stem cells , which were subsequently reinfused back into the two young patients who were enrolled in this trial [ 3 , 4 , 9 , 10 ] . the other clinical study used a retroviral - encoded neomycin resistance gene to trace autologous human tumor - infiltrating t lymphocytes in five patients with advanced melanoma [ 3 , 4 , 810 ] . moreover , a long - term clinical benefit was reported in the two young patients who participated in the first human gene therapy clinical trial for the treatment of ada deficiency . the functions of the immune system were restored in both patients [ 3 , 4 , 9 , 10 ] . remarkably , the expression of the recombinant ada gene was observed in 20% of lymphocytes of one patient ten years after the last infusion of transduced bone - marrow - derived hematopoietic stem cells . naturally , this initial success prompted for the worldwide submission of hundreds of phase i and phase ii human gene therapy protocols , which utilized various viral and nonviral gene delivery models for the treatment of monogenic disorders and cancer [ 3 , 4 ] . however , the majority of these clinical trials provided disappointing results , as the design of the various gene transfer systems were not sufficiently adequate to support efficacious human gene therapy protocols in the clinical setting [ 3 , 4 ] . common problems were related to transgene silencing following the genetic manipulation of target cells and/or mediocre transduction efficiency [ 3 , 4 ] . in addition , host immune responses to the vector systems and/or transduced cell populations constituted a very critical issue both in terms of safety and efficacy for the gene - based interventions [ 3 , 4 , 1115 , 17 ] . a young patient affected by a partial ornithine transcarbamylase deficiency died because of an acute inflammatory reaction , which was caused by a massive intrahepatic infusion of adenoviral vector particles carrying the functional copy of the defective gene [ 3 , 13 , 18 ] . this gene - based clinical trial was conducted in the united states of america in september 1999 and represented the first severe setback for gene therapy programs , which underwent austere public scrutiny [ 3 , 13 , 18 ] . therefore , the field of vector design had to tackle a variety of critical issues to control host humoral and/or cytotoxic t cell ( ctl ) immune responses against viral vector particles and/or transduced cell populations [ 3 , 4 , 1115 , 17 ] . furthermore , it was necessary to improve the production of viral and non - viral vector stocks , enhance the efficiency of ex vivo and in vivo gene transduction of several cell types , optimize transgene expression levels after gene transduction , and stabilize the duration of transgene expression in target cells [ 3 , 4 , 1115 , 17 ] . on these grounds , viral promoters were genetically modified in order to minimize de novo methylation of cpg - rich islands , which is one of the factors that are responsible for transgene silencing [ 3 , 4 , 1115 , 17 ] . stronger enhancers were utilized to sustain higher levels of transgene expression in transduced cell populations [ 3 , 4 , 1115 , 17 ] . lastly , protocols for the in vitro gene transduction of human hematopoietic stem cells were optimized [ 1117 ] . these strategies contributed to the development of more efficient vector systems for gene transfer modalities both in human gene therapy protocols and preclinical studies [ 1117 ] . optimized gene transfer protocols were utilized in the field of stem cell research for the genetic manipulation of human and mouse embryonic stem ( es ) cells , several types of adult stem cells , and , more recently , the production of induced pluripotent stem ( ips ) cells , which may derive from every type of either human or animal somatic cell [ 1933 ] . indeed , gene transfer technology has emerged as a very useful tool to support stem cell research over the last decade [ 1832 ] . the merging of gene transfer technology and stem cell research may have important implications in the field of regenerative medicine , which is aiming at developing novel therapeutic approaches for the treatment of neurodegenerative diseases , diabetes , and cardiovascular disorders [ 1933 ] . so far , the combination between gene therapy and stem cell research was applied for the treatment of hematological maladies that derive from genetic disorders , such as ada deficiency and severe combined immunodeficiency ( scid)-x1 [ 14 ] . on one hand , the integration of the shuttle vector within the chromosomal dna of the target cell is one of the requirements for a long - term transgene expression . on the other hand , however , integrative gene transfer models have a potentially dangerous downside , as they are associated with the risk of insertional mutagenesis , which may eventually result in the development of malignancies [ 3 , 4 , 11 , 12 , 14 , 15 , 17 ] . the integration of the shuttle vector may tamper with the natural chromosomal arrangement of the target cell and trigger a variety of events leading to the establishment of a transformed cell phenotype [ 3 , 4 ] . unfortunately , retroviral - mediated gene transfer was responsible for the development of leukemia in five young subjects who participated in clinical trials for the treatment of scid - x1 in france and in the united kingdom [ 1 , 2 , 3444 ] . the various outcomes reported in the scid - x1 clinical trials and the mechanisms leading to the development of insertional mutagenesis - induced carcinogenesis will be discussed in greater detail in the next section . this paper will also describe the current strategies adopted to minimize and possibly prevent insertional mutagenesis - related events in patients . scid - x1 is an inherited genetic disorder that leads to immunodeficiency and is associated with the loss of the common  chain ( c ) cytokine receptor subunit [ 36 , 45 , 46 ] . the absence of the c subunit impairs the biological and biochemical functions of the cellular receptors that bind a variety of interleukins , such as il-2 , il-4 , il-7 , il-9 , il-15 , and il-21 [ 45 , 46 ] . the impaired cellular receptors fail to interact with a variety of interleukins , which are important regulators of cell proliferation , growth , survival , and differentiation [ 42 , 43 ] . for this reason , early lymphoid progenitors become unresponsive to the previously mentioned interleukins and , for this reason , they are not able to differentiate into b , t , and natural killer ( nk ) cells [ 45 , 46 ] . the deprivation of these three cell types in the hematopoietic compartment results in a severe host immunodeficiency [ 45 , 46 ] . a retroviral - encoded c subunit was used in the various gene therapy clinical trials for the treatment of scid - x1 , which were initially conducted in france   and then in the united kingdom [ 42 , 47 ] , australia , and united states of america . beneficial clinical outcomes were reported in all of these gene - based trials [ 42 , 4750 ] . autologous cd34 bone marrow - derived cells were extracted from the patients and transduced in vitro with a retroviral - based vector system . the retroviral vector transduction was very efficient and the expression of the recombinant c subunit rendered autologous cd34 bone - marrow cells susceptible to the influence of il-2 , il-4 , il-7 , il-9 , il-15 , and il-21 . the genetically modified autologous bone - marrow cells were reinfused into the patients , in the attempt to correct the phenotype of the disease . the french clinical trial was conducted in 2000 and initially reported a considerable success , as the immune system functions were restored in 9 out of 10 patients . however , four of these patients developed a leukemia - like illness in the following months [ 13 , 3444 ] . the hematological malignancy was first observed in two patients after periods of 30 and 34 months [ 13 , 37 , 43 ] , whereas the other two patients came down with leukemia in subsequent months [ 2 , 34 , 36 , 43 ] . sadly , one of the first two patients who developed leukemia had a relapse of the disease and died in october 2004 [ 2 , 38 , 39 ] . an additional fifth case of leukemia was reported in a child who participated in the british scid - x1 trial that was conducted in 2005 . the british and the australian gene therapy clinical trials utilized lower amounts of transduced autologous cd34 hematopoietic stem cells for infusion into the patients , with the intent to reduce the risk of insertional mutagenesis - induced malignancies [ 42 , 47 , 48 ] . unfortunately , this strategy was not particularly effective , as shown by the fifth case of leukemia reported in the british scid - x1 clinical trial . there are two main mechanisms leading to insertional mutagenesis for retroviridae - based gene delivery models , which comprise retroviral- and lentiviral - derived vector systems [ 3 , 4 , 11 , 15 , 51 ] . the genera of retroviridae have two long terminal repeats ( ltrs ) situated at the 5- and 3-end of their genome ( figure 1 ) . consequently , following the integration of the retroviridae - based vector into the cellular chromosomes , the 3-ltr might promote the expression of an endogenous oncogene if this should be situated in the proximity of the integration site [ 3 , 4 , 11 , 15 ] . furthermore , the integration of the retroviridae - based vector might silence the expression of cellular tumor suppressor genes if the viral vector integration should occur within an exon of a certain tumor suppressor gene ( figure 2 ) [ 3 , 4 , 11 , 15 ] . this first mechanism that may activate the expression of cellular oncogenes and/or silence tumor suppressor genes has a short range of action . a second mechanism for insertional mutagenesis with a much longer range of action envisions the interaction between the enhancer element of a retroviral ltr and a cellular promoter driving the expression of an endogenous oncogene ( figure 3 ) [ 3 , 4 , 11 , 15 ] . the second mechanism was responsible for the onset of hematological malignancies in five patients of the scid - x1 gene therapy clinical trials [ 1 , 2 , 3444 ] . the analysis of malignant cells obtained from the first two leukemic patients of the french scid - x1 clinical trial showed an overexpression of the lim only protein 2 ( lmo2 ) oncogene [ 37 , 46 ] . lmo2 at physiological levels is an important regulator of hematopoietic stem cell development , whereas aberrant levels of lmo2 expression in t cells are associated with acute lymphoblastic leukemia [ 46 , 5254 ] , whereas the possible involvement of lmo2 overexpression in b - cell acute lymphoblastic leukemia is currently under investigation . a report analyzed the malignant t cells obtained from the first two patients who developed leukemia in the french gene therapy clinical trial for the treatment of scid - x1 . in one patient , the site of integration of the retroviral vector was 3  kb upstream of the lmo2 transcription start , whereas in the other patient the integration site was in antisense orientation to the lmo2 promoter and 5  kb downstream of the lmo2 transcription site , which corresponded to the first lmo2 intron . this study did not find any replication - competent retrovirus and ruled out a possible contribution of the overexpressed recombinant c subunit to the onset of the hematological malignancy . according to a couple of reports , an overexpressed recombinant c subunit induced tumors in animal models [ 56 , 57 ] . however , further studies on human cells excluded the possibility that the overexpressed recombinant c subunit had any carcinogenic effects [ 43 , 5860 ] . these findings , taken together , indicate that animal models do not always have the ability to recapitulate human maladies , especially in the case of oncological diseases [ 6163 ] . lmo2 overexpression , per se , might not be sufficient to promote carcinogenesis , which requires a multistep mechanism that involves a variety of genetic alterations , epigenetic mutations , activation of cellular oncogenes , and/or inactivation of putative tumor suppressor genes and environmental factors [ 6172 ] . a subsequent study analyzed the phenotype of malignant cells of the other two leukemic patients who participated in the french scid - x1 clinical trial . moreover , a second retroviral vector integration site was detected in the proximity of the proto - oncogene ccdn2 in leukemic cells of one patient , whereas malignant cells of the other patient carried an integrated retroviral vector close to the proto - oncogene bmi1 . blast cells of both leukemic patients exhibited a variety of genetic alterations , such as deletion of tumor suppressor gene cyclin - dependent kinase 2a ( cdkn2a ) , chromosomal translocations , sil - tal1 rearrangements , 6q interstitial losses , and gain - of - functions resulting in the activation of notch1 . almost analogous findings were reported in the phenotypic analysis of blast cells of the leukemic patient who was enrolled in the british scid - x1 gene therapy clinical trial . lmo2 overexpression was observed also in this case , along with a variety of genetic aberrations , which included loss of expression of the tumor suppressor gene cdkn2a , gain - of - function of notch1 , and translocation of the t - cell receptor ( tcr)-b region to the stil - tal1 locus . a protocol based on linear amplification - mediated pcr ( lam - pcr ) was developed to analyze the retroviral vector integration sites within the human genome following a gene - based intervention in patients [ 7376 ] . the lam - pcr protocol was used to determine the integration site profile of retroviral - based vectors within the genome of cd34 hematopoietic stem cells of two patients with wiskott - aldrich syndrome ( was ) , who participated in a phase i gene therapy clinical trial [ 7678 ] . was is a genetic disorder characterized by micro - thrombocytopenia , frequent infections , eczema and is associated with a high incidence of lymphoreticular malignancy and autoimmune disorders . the retroviral vector system was utilized to express the functional copy of the human was gene in autologous cd34 hematopoietic stem cells , which were transduced ex vivo and then injected back into the patients [ 76 , 77 ] . lam - pcr analysis was conducted 892 and 891 days after intervention in patients 1 and 2 , respectively . this analysis reported 5,709 and 9,538 unique retroviral vector integration sites in patients 1 and 2 , respectively . the most recurrent common integration sites ( cis ) involved the following genetic loci : lmo2 , mds - evi1 , ccdn2 and prdm16 [ 76 , 77 ] . retroviral integration sites either in the proximity or inside the lmo2 and ccdn2 gene loci were more frequent in lymphoid cells , whereas retroviral integration sites involving either the prdm16 and mds1-evi1 gene loci were predominant in myeloid cells [ 76 , 77 ] . the reconstitution of the hematopoietic compartment in both patients exhibited polyclonal blood cell populations , which indicates the absence of hematological malignancies [ 76 , 77 ] . further studies are currently in progress in order to better characterize the mechanism of insertional mutagenesis - induced hematological malignancies in patients . indeed , the characterization of retroviral vector integration sites in dominating monoclonal blood cell population might reveal important clues on the deregulated cellular signaling systems , which may play a role in the establishment of a malignant cell phenotype and impart clonal expansion . the use of retroviridae - derived vector systems in clinical trials poses a serious safety concern , because of the onset of hematological malignancies that may be promoted by insertional mutagenesis events [ 13 ] . as already mentioned , insertional mutagenesis - induced malignancies were reported in five leukemic patients of the scid - x1 gene therapy clinical trials [ 13 , 3444 , 51 , 81 ] . insertional mutagenesis events were also observed both in animal systems [ 44 , 58 , 8284 ] and human cell culture models [ 8491 ] . these preclinical studies focused on the integrating properties of retroviridae - derived vector systems based on murine leukemia virus ( mlv ) , avian sarcoma - leukosis virus ( aslv ) , and human immunodeficiency virus type 1 ( hiv-1 ) [ 8592 ] . the integration of the aforementioned retroviridae - derived vector systems was not completely random within the human genome . in fact , most of integration sites were preferentially localized in the proximity of chromosomal regions that contained transcriptionally active genes [ 8591 ] . in addition , mlv - derived vector systems showed a remarkable predisposition for integration points near transcription start sites [ 85 , 86 ] , whereas hiv-1-based lentiviral vector systems favored genomic loci rich in active genes and intercalated with chromosomal regions containing methylated cpg islands that are not permissive for gene expression [ 8589 ] . in contrast , aslv - based vector systems did not exhibit any specific preference for transcription start sites and had no bias for regions containing active genes .    all integrating gene delivery models may present the risk of insertional mutagenesis in target cells . in fact , experiments in animal systems and cell culture models demonstrated that insertional mutagenesis might also be triggered by aav - based vectors   and integrating non - viral vector systems , such as sb dna transposon - derived vectors . in vivo administration of aav - derived vectors was responsible for either insertional mutagenesis - induced angiosarcomas or hepatocellular carcinomas in mice [ 12 , 92 ] . other studies on human cell culture systems detected residual promoter - like properties in terminal repeat sequences of sb dna transposon - derived vectors , which was able to induce the expression of cellular genes that were contiguous to the integration site of the non - viral vector system . many studies placed an emphasis on integration of retroviral- and aav - derived vector systems within common fragile sites of the human genome [ 90 , 9496 ] . human common fragile sites consist of specific regions linked with chromosomal breakpoints , which may play a relevant role in the early stages of malignant cell transformation [ 95 , 97102 ] . interestingly , a number of human oncogenic viruses integrate preferentially their genome within common fragile sites . these human oncogenic viruses comprise human papilloma virus [ 103106 ] , epstein - barr virus [ 95 , 107 , 108 ] , and hepatitis b virus . the analysis of so - called hotspots for certain viral - derived vector systems integration sites is essential to determine the mechanism of insertional mutagenesis - related onset of malignancies in patients . gene therapists are currently addressing the issue of insertional mutagenesis - induced malignancies in patients by pursuing three main approaches : ( i ) engineering of safer integrating gene delivery models ; ( ii ) development of episomal gene delivery systems with improved duration of transgene expression in transduced cell populations ; ( iii ) production of genetically modified meganucleases and zinc - finger proteins for the correction of genetic defects . the central issue for the development of safer integrating gene delivery systems consists of producing gene transfer shuttle vectors that do not affect the physiological genomic organization of transduced cells . therefore , it is necessary to prevent interactions between enhancer and/or promoters of the vector system and the host genome . the engineering of self - inactivating retroviridae - derived vectors may minimize the incidence of insertional mutagenesis - related events in transduced cells [ 3 , 11 , 15 ] . the replication cycle of the genera of retroviridae comprises an initial transcription stage , in which the viral messenger rna ( mrna ) is synthetized and subsequently packaged into the virion ( figure 4 ) . this viral mrna does not contain the enhancer / promoter u3 region in the 5-ltr and the u5 region in the 3-ltr . after cell infection , the viral mrna is released into the cytoplasm and then is reversed transcribed into double stranded proviral dna . at this stage , the u3 region in the 3-ltr is copied at the 5-ltr , whereas the u5 region in the 5-ltr is copied at the 3-ltr . the double - stranded proviral dna is successively assembled in a preintegration complex , which crosses the nuclear membrane and is integrated within the genome of the infected cell ( figure 4 ) [ 3 , 11 , 15 ] . self - inactivating retroviridae - derived vectors are produced by a deletion of the u3 region in the 3-ltr ( figure 5 ) [ 3 , 11 , 15 ] . , the 5-ltr drives the transcription of a viral mrna lacking the u3 region in the 3-ltr . following the reverse transcription process in transduced cells , the truncated u3 region in the 3-ltr is reproduced at the 5-ltr . this results in a proviral dna without transcriptional activity at both ltrs ( figure 5 ) . obviously , self - inactivating retroviridae - derived vectors need an internal promoter to express the transgene ( figure 5 ) [ 3 , 11 , 15 ] . self - inactivating retroviridae - derived vectors may be considered safer than their original counterparts , which are termed gamma - retroviral or gamma - lentiviral based vectors [ 3 , 11 , 15 ] . the deletion of retroviral enhancers and promoter regions in the ltrs minimizes the probability of interactions between the transfer vector system and the host genome [ 3 , 11 , 15 ] . in fact , the nontranscribing 3-ltr is not able to drive the expression of cellular oncogenes that might be present in the vicinity of the integration site ( figure 5 ) . moreover , the removal of retroviridae enhancer regions precludes the long - range interference with cellular promoters that control the expression of endogenous oncogenes ( figure 5 ) [ 3 , 11 , 15 ] . however , the presence of an enhancer within the internal promoter of a self - inactivating retroviridae - derived vector still poses the issue of insertional mutagenesis , as it may stimulate cellular promoters of oncogenic factors [ 3 , 11 , 15 , 110 , 111 ] . studies produced self - inactivating lentiviral vectors containing enhancer - less internal promoters [ 110 , 111 ] . in one case , a self - inactivating lentiviral vector system utilized as internal promoter the enhancer - less and methylation - free cpg islands promoter of the ubiquitously acting chromatin opening elements , which is a human housekeeping gene and was termed a2ucoe [ 110 , 112 ] . the use of the a2ucoe internal promoter has a dual advantage : the first advantage is related to the vector design safety improvement that is due to the absence of enhancer regions ; the second advantage is correlated with the absence of cpg - rich islands within the sequence of the human housekeeping gene promoter , which confers resistance to de novo methylation - mediated transcriptional silencing [ 110 , 112 ] . the self - inactivating retroviridae - derived vector containing the enhancer - less a2ucoe internal promoter was utilized for the gene transduction of the recombinant c subunit in primary bone - marrow - derived cd34 hematopoietic stem cells of a patient with scid - x1 . transduced cell populations readily expressed the recombinant c subunit and became susceptible to the action of interleukins in the in vitro system . these findings indicate that the production of enhancer - less self - inactivating retroviridae - derived vectors constitute a significant improvement in safety design .    in other studies , the internal promoters of self - inactivating lentiviral vector systems were based either on the enhancer - less human vav1 promoter [ 111 , 113 ] or the phosphoglycerate kinase ( pgk ) promoter . the enhancer - less human vav1 promoter was used for the expression of the recombinant c subunit in a murine model of scid - x1 . although the transgene expression levels were not high , it was possible to correct the phenotype of the disease in a mouse model . another preclinical study on fanconi anemia utilized the enhancer - less human vav1 promoter and the phosphoglycerate kinase ( pgk ) promoter . fanconi anemia is an inherited genetic disorder leading to bone marrow failure and high incidence of leukemia . genotypic analysis of patients with fanconi anemia identified mutations in a group of fifteen genes , which were termed fanc genes . mutations in the so - called fanca gene are among the most frequent in patients with fanconi anemia . self - inactivating lentiviral vector system was utilized in a preclinical study to express the functional copy of fanca , which was driven either by the enhancer - less human vav1 promoter or the phosphoglycerate kinase ( pgk ) promoter . however , the levels of transgene expression were rather low . in order to optimize fanca expression levels , the investigators utilized a variant of the woodchuck hepatitis virus posttranscriptional regulatory element ( wpre ) region . overall , all tested vector systems induced a comparable phenotypic correction in cell culture models for fanconi anemia . a self - inactivating lentiviral vector was utilized in a mouse model for the correction of scid - x1 . this vector system used a minimal promoter obtained from the eukaryotic elongation factor  ( ef1 ) gene to drive the expression of a codon - optimized human c cdna . remarkably , this vector system was able to correct the phenotype of the disease in the mouse model , without causing the overexpression of the lmo2 oncogene . in contrast , the self - inactivating lentiviral vector system containing the human c promoter driving the expression of the human c cdna induced the over - expression of lmo2 . the chicken hypersensitivity site 4 ( chs4 ) insulator was placed in the 3-ltr of both vector systems . insulators were utilized to prevent the silencing of retroviridae - encoded transgenes , which may derive either from de novo methylation of viral promoters and/or position effects imparted by chromosomal sequences surrounding the retroviral integration site [ 116 , 117 ] . however , the result of this study indicates that insulators are not efficacious in providing a barrier against the genotoxicity of retroviridae - based vectors . studies are currently in progress in order to identify novel insulators that have the ability to prevent genotoxic effects of integrating gene delivery systems . a major inadequacy of self - inactivating retroviridae - derived vectors consists of an increased polyadenylation ( polya ) signal read - through in the 3-ltr ( figure 6 ) . leaky transcriptional termination signals may result in the activation of silent cellular oncogenes in transduced cells ( figure 6 ) . furthermore , the polya read - through in packaging cell lines might cause the uptake of cellular oncogenes into the genome of retroviridae - based vectors , which , in turn , may transmit it to transduced cell populations . the deletion of the u3 region from the 3-ltr enhances the probability of polya read - through in self - inactivating retroviridae - based vectors [ 123 , 124 ] . in fact , the u3 region of the ltr contains both transcription termination motifs and enhancer - promoter elements [ 123 , 124 ] . in this respect , a study demonstrated that the incorporation of seven sv40-derived upstream polya enhancer elements in the residual u3 region improved transcription termination efficiency in self - inactivating retroviridae - based vectors . this study reported also a 3-fold enhancement in viral vector titers , along with increased and stable transgene expression levels in transduced cell populations . insertional mutagenesis - induced malignancies were also observed in animal models for aav - mediated gene transfer . this safe integration site is termed aav1 and is located in the q arm of the chromosome 19 , between q13 - 3 and qter [ 3 , 12 ] . the wild - type aav - specific integration into aav1 is mediated by the viral factors rep 68 and rep 78 [ 3 , 12 ] . however , most of the wild - type aav genome must be removed in order to engineer aav - derived vector systems , because of the limited capacity of aav - based vectors in accommodating transgenes [ 3 , 4 , 12 ] . for this reason , aav - derived vectors do not have rep 68 and rep 78 proteins and , therefore , integrate randomly within the cellular genome [ 3 , 4 , 12 ] . the field of vector design is attempting to restore aav1-specific integration via coexpression in transduced cell populations of aav - based vectors along with rep 68 and rep 78 proteins . another strategy to circumvent the issue of insertional mutagenesis consists of producing episomal gene delivery models with enhanced duration of transgene expression . on one hand , episomal vector systems do not alter the transduced cell genome , as they do not integrate into the chromosomal dna . on the other hand , nonintegrating gene delivery models only allow for a transient expression of the transgene . on these grounds , the field of vector design is working on the development of novel systems for the stabilization of transgene expression after the episomal vector - mediated gene transfer into target cells . to this end , gene therapists placed a particular emphasis on the development on episomal lentiviral vectors [ 125135 ] , because of their ability to transduce proficiently an ample variety of mammalian cells and regardless of their cell cycle phase . several studies showed that episomal lentiviral - based vectors maintained high transduction efficacy and wide cell tropism [ 125 , 128131 ] . studies on mouse models focused on in vivo gene delivery of episomal lentiviral vectors in the central nervous system , stem cells of the hematopoietic compartment , muscles , and ocular tissues . episomal lentiviral vector systems can be produced via inactivation of the viral integrase and mutation of the integrase attachment sites ( att ) that are present in the u3 region of the 5- and 3-ltrs . the viral integrase is inactivated by site directed mutagenesis of the chromosome - binding moiety , proviral dna binding domain and catalytic site [ 125 , 130 ] . these point mutations do not affect at all the ability of the viral integrase to transport the pre - integration complex through the nuclear membrane [ 125 , 130 ] . negligible levels of residual integration of episomal lentiviral vectors were reported in some transduced cell populations . nevertheless , such a residual integration activity was related to background recombination events , rather than the mutated viral integrase . episomal lentiviral vector systems allow for a better long - term transgene expression in nondividing cells than in dividing cells , as mitosis may cause the dilution of the episomal vector genome in progeny cells [ 125 , 128 , 131 ] . remarkably , the inclusion of scaffold / matrix attachment regions ( s / mar ) may increase the duration of transgene expression of episomal lentiviral vector systems within transduced cell populations [ 136 , 137 ] . this approach may also be utilized to enhance the episomal permanence of non - viral gene - delivery models in transfected human cells [ 137 , 138 ] . the efficacy of s / mar in increasing episomal permanence was cell - type - dependent . further research is necessary to improve the preclinical applications of nonintegrating gene delivery models . meganucleases and artificially engineered zinc - finger proteins are attracting a great deal of interest in the field of gene transfer technology , as they hold the potential of conducting either site - specific rectification of defective genes , or inclusion of genetic elements into selected loci within the genome of transfected cells [ 139143 ] . the c2h2-type zfps are by far the most common dna - binding moieties of transcription factors . amazingly , such dna - binding domains constitute about 2% of the entire human genome [ 139144 ] . this strategy relies on the variation of c2h2-type zfps that are present in the sp1 transcription factor . a similar tactic was utilized for the production of recombinant meganucleases or homing endonucleases [ 143 , 145148 ] . meganucleases consist of sequence - specific endonucleases that identify relatively large dna binding motifs in living cells . such dna binding motifs are in the range of 14 base pairs ( bp ) or more . the production of genetically modified meganucleases might allow for the design of site - specific gene delivery into the human genome [ 145148 ] . for example , the i - crei - derived meganuclease dna binding domain was adjusted to recognize specifically new designed genetic sequences [ 145147 ] . the artificial i - crei - derived meganuclease was utilized to target the human xeroderma pigmentosum group c ( xpc ) gene , whose genetic mutations may be responsible for the pathogenesis of xeroderma pigmentosa disease , which is a rare autosomal recessive genetic illness causing both hypersensitivity to ultraviolet light and high incidence of skin cancer . two independent reports showed an efficient i - crei - derived meganuclease - mediated rectification of a flawed chromosomal locus in various mammalian cell lines [ 146 , 148 ] . interestingly , the employment of genetically engineered meganucleases was not associated with noticeable levels of genotoxicity in mammalian cell lines [ 143 , 148 ] . other studies are currently underway for the characterization of i - scei [ 150152 ] and i - dmoi meganucleases . the possible onset of insertional mutagenesis - induced malignancies in patients constitutes a serious obstruction to the establishment of gene therapy programs in the clinical setting . undeniably , gene transfer technology holds enormous therapeutic potential for the treatment of a wide variety of pathological conditions , such as cancer , cardiovascular disorders , genetic diseases , diabetes , neurodegenerative illnesses , and infectious maladies [ 3 , 4 ] . moreover , the merging between gene transfer technology and stem cell research may have important implications in the area of regenerative medicine . the field of vector design is currently tackling the critical issue of insertional mutagenesis by adopting three main strategies . the first approach is based on the production of integrating gene delivery models that can only express the transgene and do not interact with the human genome , in order to avoid the activation of cellular oncogenes . in this respect , enhancer - less self - inactivating retroviridae - derived vectors have attracted a lot of interest and seem very promising . the second approach depends on the engineering of episomal vector systems , which were genetically modified to allow for a more stable and long - lasting transgene expression in transduced cell populations . the third approach utilizes genetically modified meganucleases and artificially engineered zinc - finger proteins to carry out either site - specific correction of faulty genes or inclusion of genetic factors into specific and safe loci of the genome of transfected cells . however , the solution of the insertional mutagenesis issue may bring a substantial contribution to a more successful application of gene therapy programs in various sectors of experimental medicine .
integrating gene delivery systems allow for a more stable transgene expression in mammalian cells than the episomal ones . however , the integration of the shuttle vector within the cellular chromosomal dna is associated with the risk of insertional mutagenesis , which , in turn , may cause malignant cell transformation . 
 the use of a retroviral - derived vector system was responsible for the development of leukemia in five children , who participated in various clinical trials for the treatment of severe combined immunodeficiency ( scid - x1 ) in france and in the united kingdom . 
 unfortunately , the hematological malignancy claimed the life of one patient in 2004 , who was enrolled in the french clinical trial . 
 in addition , adeno - associated - viral-(aav- ) mediated gene transfer induced tumors in animal models , whereas the sleeping beauty ( sb ) dna transposon system was associated with insertional mutagenesis events in cell culture systems . on these grounds , 
 it is necessary to develop safer gene delivery systems for the genetic manipulation of mammalian cells . 
 this paper discusses the latest achievements that have been reported in the field of vector design .