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a Kimmel Cancer Center, Jefferson Medical
College, Thomas Jefferson University, Philadelphia, Pennsylvania, USA;
b Department of Pathology, Anatomy and Cell
Biology, Jefferson Medical College, Thomas Jefferson University, and
Sbarro Institute for Cancer Research and Molecular Medicine,
Philadelphia, Pennsylvania, USA
Key Words. Gene therapy • Clinical trials • Gene delivery systems in vivo or in vitro • Retroviruses • Adenovirus • Adeno-associated virus • Cationic liposomes
Correspondence:
Dr. Gaetano Romano, Kimmel Cancer Center,
Jefferson Medical College, Thomas Jefferson University, 624 Bluemle
Life Sciences Building, 233 South Street, Philadelphia, Pennsylvania
19107, USA.
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ABSTRACT |
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 Top Abstract IntroductionGene Transfer
ModelsVector Design for In...ConclusionReferences |
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Introduction |
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TopAbstractIntroductionGene Transfer
ModelsVector Design for In...ConclusionReferences |
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Also in 1990, the first gene therapy clinical trial for the treatment of patients with melanoma [4] was conducted. The results of this study indicated that retroviral-mediated gene transfer in patients was safe. This finding prompted the submission of many other protocols for gene therapy clinical trials to treat patients affected by cancer, primarily in the area of melanoma [5-10], followed by ovarian carcinoma [11], sarcoma [10], brain tumor [12], and lung cancer [13].
There is also a strong interest in beginning gene therapy clinical trials for the treatment of patients with AIDS, cardiopathies, and neurologic diseases. Indeed, gene transfer technology has already been applied in the phase I and phase II trials for the AIDS vaccine programs, which have recently begun in the USA [14-16]. These vaccine programs are aiming at inducing both humoral and cytotoxic T lymphocyte (CTL) immune responses to HIV-1 in an attempt to eradicate the virus from the patients and to develop protective immunity to HIV-1 transmission in healthy individuals who are at risk of infection. In order to elicit CTL immune responses, the viral antigens must be intracellularly processed within target cells to express various peptidic epitopes associated with host HLA class I antigens on the cell membrane. This may be achieved by gene transfer technology, such as viral vectors carrying HIV-1 genes [14-16], or naked DNA [14, 15, 17]. Humoral immune responses are normally directed at the HIV-1 envelope, whereas HIV-1 specific CTL are usually against gag, pol, or nef [18].
To date, the viral vectors used in the AIDS vaccine programs in humans and primates are vaccinia virus and canarypox virus [14]. Other viral vectors based on Semliki Forest virus, rhinovirus, and poliovirus are currently under development [14]. Vaccinia viral vector has been engineered to deliver HIV-1 envelope (gp120 or gp160) together with the p24 subunit of gag (gag p24) [14], whereas the canarypox-based viral vector has been used to deliver only gag p24 [14]. Subunits of pol and nef have not been tested yet.
Hopefully, this innovative HIV-1 vaccine design will overcome the complex issue of viral diversity, which, besides posing a key obstacle to the development of vaccines to HIV-1 [19], displays a fundamental role in the pathogenesis of AIDS [20, 21].
There is an enormous variety of possible applications of gene transfer in therapy. As already anticipated, the spectrum ranges from the treatment of inherited or acquired genetic disorders to cancer, AIDS, cardiopathies, and neurologic diseases. This is strongly encouraging to the pursuit of gene therapy programs in medicine. However, after a first phase of enthusiastic research developments, the expectations of investigators are now more sober. Although much effort has been directed in the last decade toward improvement of protocols in human gene therapy, and in spite of many considerable achievements in basic research, the therapeutic applications of gene transfer technology still remain mostly theoretical. The weakest point of gene therapy development programs is, paradoxically, vector design, followed by gene regulation and avoidance of immune responses. Basic research is cautiously progressing to address these pressing issues. The goal of this review is to summarize the standpoint of the various basic research projects, which have been planned to improve the protocols of oligonucleotide and gene delivery in therapy.
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Gene Transfer Models |
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TopAbstractIntroductionGene Transfer
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Other viral vectors that are currently under development are based on lentiviruses [53-58], human cytomegalovirus (CMV) [59], Epstein-Barr virus (EBV) [60], poxviruses [61, 62], negative-strand RNA viruses (influenza virus) [63], alphaviruses [64], and herpesvirus saimiri [65]. Also of extreme interest is the construction of a hybrid adenoviral/retroviral vector, which has successfully been used for in vivo gene transduction [66]. The characteristics of the most developed gene delivery systems are summarized in Table 1.
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The difficult tasks of vector design have to deal with safety issues, improvement of in vivo gene delivery efficiency, and gene regulation post-cell transduction. These tasks are all related to one another. Most of the previously mentioned phase I gene therapy clinical trials for the treatment of inherited genetic diseases and cancer were carried out by ex vivo administration of retroviral vectors into target cells, which were then reimplanted into the patients (i.e., treatment of ADA-deficiency, hemophilia B, Fanconi's anemia, Gaucher's disease, Hunter syndrome, LDL-deficiency, and melanoma). In contrast, the treatment of cystic fibrosis was carried out by in vivo administration of vectors based on adenovirus, cationic liposomes, or AAV. The parameters of these in vivo administrations of vectors in clinical trials are still far from ensuring efficient therapeutic interventions. The vectors used in these studies had some positive properties and were relatively safe. As summarized in Table 1, these gene delivery systems can transduce nondividing cells, avoid cell mutagenesis due to the random transgene integration in the host chromosomal DNA (except for AAV-based vectors) and can be rather easily administered to the patients in high doses; however, they are affected by many limitations. Adenoviral vectors can elicit host immune responses and are not suitable for long-term expression of the transgene, especially in vivo. Liposome-based vectors are not infectious and have a low degree of toxicity, but they also do not allow for stable transgene expression, and their in vivo applications are difficult for a variety of reasons ( Table 1). The interest in AAV is mainly related to its property of integrating the viral genome in a safe host chromosomal site [31-35]. Unfortunately, such a property is lost in AAV recombinant vectors, and this may result in cell mutagenesis.
The field of gene therapy is now actively involved in the challenging task of improving the design of vector systems for in vivo applications.
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Vector Design for In Vivo Gene Delivery |
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TopAbstractIntroductionGene Transfer
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Each vector system has a series of advantages, problems, and preferential applications in therapy. As previously mentioned, the problems in vector design for in vivo applications are generally related to safety issues, improvement of vector production, and control of transgene expression post-cell transduction. The first rule in the matter of vector design is that the gene delivery systems must not be pathogenic or toxic to the patients. Therefore, the various viral vectors must be engineered to be noncompetent for replication and must not contain viral genes encoding for factors which may pose a hazard in humans. It has been argued whether the removal of putative virulence may be detrimental to the transduction potential. Results indicate that viral vectors so far produced retain their infectivity, although they do not replicate.
The in vivo administration of viral vectors requires additional safety regulations compared to the ex vivo one. In order to avoid the ectopic expression of the transgene, viral vectors should be engineered to have a cell tropism specific for the target cells, especially if the viral vectors can also transduce nondividing cells. In this respect, there have been many attempts, with small success, to alter the cell tropism of viruses that are nonpathogenic in humans in order to engineer chimeric viruses capable of infecting distinct human cells. These studies involved mainly recombinant retroviruses and lentiviruses and will be described in the next paragraph.
Another line of investigation is aiming at controlling in vivo transgene expression by developing vector systems containing internal tissue-specific or inducible promoters. The latter are based on: metalloprotein gene promoter, steroid or tetracycline-inducible promoters, Cre/LoxP recombination system, promoters responsive to the insect hormone ecdysone and retinoids. The in vivo regulation of transgene expression within the therapeutic window is also a very important goal that must be achieved. Unfortunately, there are many elusive problems to be solved which derive mostly from the empirical knowledge basic researchers have in this matter.
The site-specific proviral integration in the host chromosomal DNA is another strongly desired feature. Possibly, this may be accomplished by opportune rearrangement of AAV-based vectors.
Other issues that vector designers are dealing with are: avoidance of immune responses (in the case of adenoviral vectors), improvement of high-titer viral vector stocks, and purification procedures.
Some progress has been made in improving the various gene delivery systems. Their variety is too vast to be described in greater detail, therefore, only the main vector models will be reviewed.
Retroviral and Lentiviral
Vectors
Undoubtedly, retroviruses are among the most efficient
tools for gene transduction of mammalian cells. For this reason, they
were successfully used in the early gene therapy clinical trials for
the treatment of inherited genetic diseases [2, 3] and cancer [4-13]. The most common retroviral vector is based
on the amphotropic Moloney murine leukemia virus (MLV) [68]. This system is particularly
suitable for efficient in vitro cell transduction: the amphotropic MLV
has a broad cell tropism, it can be produced at relatively high titers
(106-107 iu/ml), and allows for long-term
transgene expression because of the viral integration in the host
chromosomal DNA.
Another important feature of retroviruses is that although they do not elicit immune responses in the host, they are susceptible to rapid degradation by the complement [69]. This is a major limitation for in vivo retroviral-mediated gene transfer. Optimal titers for in vivo applications should be in the range of 1010 iu/ml, whereas the maximum titer that can be obtained barely reaches 107 iu/ml. In addition, retroviral particles are difficult to concentrate, as they are fragile and can be destroyed during the precipitation. This problem can be circumvented by pseudotyping the retroviral core with the G glycoprotein of vesicular stomatitis virus (VSV G). This envelope stabilizes the retroviral particles, which can then be easily concentrated by ultracentrifugation of the retroviral supernatant [70, 71].
Retroviral stocks are mainly produced by transient expression systems [72-76], which offer a variety of advantages: the retroviral titers are in the range of 106-107 iu/ml, that are from 10- to 50-fold greater than those obtained by conventional packaging cell lines; the production of retroviral stocks is rapid and highly reproducible; the transient retroviral expression practically rules out the possibility of replication-competent virus formation. The latter feature may greatly facilitate the in vivo retroviral-mediated gene transfer.
As shown in Figure 1, the retroviral genome was divided among three plasmids. Both gag/pol and the envelope (env) are under the control of the human cytomegalovirus (CMV) promoter. The 5' and 3' long-terminal repeats (LTRs) and the packaging signal (Y) were deleted in these two constructs, therefore, the mRNA encoding for gag/pol and for env is the only substrate for translation in the transfected cells. The retroviral-transfer vector has the two LTRs and the packaging signal (Y) and encodes for a chimeric gene whose mRNA can be packaged into the virion and reverse-transcribed in the target cells' cytoplasm; the resulting cDNA is then delivered to the cell nucleus and integrated into the host genome. The chimeric gene may be a therapeutic factor and/or a reporter gene. The production of high-titer retroviral stocks is carried out by transient cotransfection of the three plasmids (gag/pol, env, and transfer vector) in highly transfectable cell lines that express the SV40 large T antigen [73]. The plasmids containing the gag/pol and env cassettes carry the SV40 origin of replication in their backbone. Therefore, post-cell cotransfection, the plasmids' copy number is greatly enhanced by the SV40 large T antigen [75]. The high DNA copy number and the massive production of gag/pol and env by the strong human CMV promoter result in an optimized retroviral titer [73, 75]. The recombinant retroviral vector was engineered to sustain a single round of infection, and the fact that the proviral genome was divided among three plasmids rules out the possibility of replication-competent virus formation by homologous recombination [75].
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Retroviral transfer vectors have also been designed to deliver transgenes under the control of internal inducible or tissue-specific promoters [78, 79]. The presence of an extra internal promoter may interfere with the 5' LTR transcriptional activity, and/or vice versa [79]. For this reason, the retroviral vectors were engineered to have an active 5' LTR in the proviral form, which is then disactivated after the viral genome integration in the host chromosomal DNA. This may easily be achieved by performing a small deletion in the 3' LTR of the proviral transfer vector [78]. Such retroviral vectors have been named self-inactivated vectors (SIV) [78].
Another important line of investigation is considering the engineering of chimeric retroviruses with specific cell tropism. This would greatly facilitate the in vivo application of retroviral vectors in clinical trials. In this respect, there have been many attempts to alter the cell tropism of ecotropic retroviruses, which do not infect human cells. This approach consists of placing foreign genes into the retroviral envelope in order to confer a cell tropism specific for certain human cell types. The foreign genes used in the early studies to generate hybrid envelopes were: CD4 [80, 81], single-chain antibodies [82-84], the polypeptide erythropoietin [85], short peptides binding to several integrins [86], and human heregulin [87]. The retroviral systems used in these studies were: avian leukosis virus [80, 86], ecotropic MLV [81, 82, 85, 87], spleen necrosis virus [83, 84], and amphotropic MLV [88]. In some cases, there has been a partial success in redirecting the cell tropism of ecotropic retroviruses [81, 83-88], but the transduction efficiency is far from being optimal for in vivo applications. A number of more recent reports have shown some improvement of transduction efficiency by chimeric viral particles with altered cell tropism [89-91]. The viral vectors used in these studies were based on adenovirus [89, 90] and on Sindbis virus [91]. Interestingly, two other groups of investigators have engineered chimeric rabies virus [92] and VSV [93], which were pseudotyped with CD4- and CXCR4-derived proteins. The latter is the coreceptor for T cell tropic HIV-1 strains [94, 95]. These studies showed that both chimeric viruses selectively infected and induced cytopathic effects in cultured cells harboring HIV-1 [92, 93]. This finding is certainly a leap forward from the preliminary study conducted by Young et al. [80]. However, it remains to be confirmed whether these chimeric viruses will be able to seek out and selectively destroy HIV-1 infected cells in the in vivo model.
An important property of retroviruses is that they can only infect actively dividing cells [67], as the transport of the preintegration complex to the nucleoplasm requires the breakdown of the nuclear membrane. Conversely, lentiviruses, such as HIV-1, also have the capability of infecting nondividing cells [96-98]. The requirement for cell division for retroviral infection has relevant implications in gene transfer technology. A positive aspect is that in vivo retroviral-mediated gene delivery in cancer therapy is facilitated because of the specific gene targeting of neoplastic cells over normal tissues. On the other hand, the lack of retroviral infection of nondividing cells precludes their in vivo gene transfer applications for neurons, hepatocytes, myofibers, and hematopoietic cells. In this prospective, the engineering of HIV-based lentiviral vectors will prove very useful. Many nonproliferating cell lines can be easily manipulated with this HIV-based vector system to generate cell culture models that stably express transduced genes. Preliminary in vitro experiments indicated that terminal differentiated neurons [99] and terminal differentiated macrophages [57] were efficiently transduced, and the reporter gene expression was stable. This finding mirrors that of another in vivo study, in which a lentiviral vector carrying a reporter gene was injected into adult rats' brains, in order to transduce neurons [53, 54]. In this case too, efficient gene delivery and a stable expression of the transgene were observed. The lentiviral-based vector systems are most likely going to implement the therapeutic efficiency of gene transfer technology in the near future. Before then, the lentiviral vectors must be thoroughly tested for biological safety. The possible reconstitution of pathogenic replication-competent HIV-1 must be excluded. The lentiviral vector stocks are also generated by transient overexpression systems [73-76], in which the packaging components (gag/pol and env) have been placed on two different plasmids and are under the control of the human CMV promoter, and the transfer vector is on a third plasmid [53]. Furthermore, the HIV-1 envelope has been deleted in this system, to be replaced by the amphotropic MLV or VSV G envelopes [53]. The HIV-1 genome has six additional reading frames to the prototypic gag, pol, and env genes that are common to all retroviruses ( Fig. 2). These extra six reading frames encode for the following factors: tat, rev, vif, vpr, vpu, and nef. Viral replication is mediated by the so-called regulatory tat and rev proteins, which respectively control viral transcriptional and post-transcriptional pathways. The other four factors (vif, vpr, vpu and nef) are called "accessory proteins" [16]. The function of these accessory proteins in HIV-1 pathogenicity is very complex and not completely understood. They are essential to maintain virulence in vivo [100] and may interfere with the cell cycle and/or cell growth [16, 100]. Their presence may per se represent a hazard in humans, regardless of the lack of HIV-1 infection. In two latest reports, the accessory proteins were deleted from the lentiviral vector system without compromising their transduction efficiency [57, 58]. This is another substantial step forward in the development of a safer lentiviral vector system. There are still many other aspects of lentiviral's biology that have to be investigated prior to considering their application as vectors in clinical trials. The main concern is about possible cell cycle and/or cell growth dysregulations by tat protein, and the random proviral integration in the host genome, which may result in mutagenesis. This phenomenon may be more dramatic for in vivo applications of lentiviral vectors than for retroviral-mediated gene transfer because of the capability of lentiviruses to also infect nondividing cells. This may predispose the lentiviral-based vectors in delivering and inserting the transgene into the genome of wrong cell types or tissues, provoking possible harm to the patients.
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Adenoviral
Vectors
Adenoviruses, together with retroviruses, constitute
the most advanced gene therapy forefront of the basic research
development for gene delivery systems.
Adenoviruses are large non-enveloped DNA viruses with a double stranded genome of 36 kb and a capsid diameter ranging from 65 nm to 80 nm [38, 39]. So far, 49 serotypes of human adenoviruses have been identified and classified into six groups according to similarities in their genome organization and hemagglutinin activity. The diameter of the viral particles depends on the serotype. Human adenovirus was isolated for the first time in 1953, when a spontaneous in vitro culture degeneration of some adenoidal tissues was observed [37]. Later, it was found that the etiologic agent responsible for this cytopathic effect was a virus, which was the reason for its being named "adenovirus" [101]. The various adenoviral serotypes can be found in distinct tissues, such as the upper respiratory tract, the conjunctiva, and the intestines.
The first recombinant adenoviral vectors were engineered in 1985 and were based on the serotypes 2 and 5 [40-42]; they are not associated with severe diseases and do not cause tumors in animals, in contrast to the other serotypes. The first adenoviral-mediated gene transfer applications in clinical trials were carried out at the beginning of the 1990s for the treatment of patients affected by cystic fibrosis [102]. Probably, adenoviral vectors will also be employed soon in cancer therapy and in the treatment of familial hypercholesterolemia and neurological and cardiovascular disorders. Many in vitro and in vivo studies in animal models have already been performed along these lines of research [103-107]. As anticipated, adenoviruses are highly immunogenic and may originate inflammatory and toxic reactions in the host [108, 109]. This poses a severe limitation to the possible applications of adenoviral-mediated gene transfer for the treatment of hereditary disorders, cardiopathies, and neurologic diseases. In addition, in all these illnesses, long-term transgene expression is required. Adenoviral vectors only allow for transient expression, because the adenoviral genome is extrachromosomal in the infected cell.
On the other hand, adenoviral-mediated gene transfer offers some advantages over retroviral vectors. First of all, adenoviral vectors can be produced at very high titers (1010 pfu/ml), which can be easily concentrated to 1012 pfu/ml. The adenovirus has the capability of encapsulating DNA molecules up to 6% bigger than the wild-type viral genome; therefore, 7-8 kb DNA inserts can be introduced in the vector. Theoretically, it may be possible to introduce in the virion much bigger DNA fragments than 7-8 kb, providing that the adenovirus genome is properly deleted. Adenoviruses can also infect nondividing cells, in contrast to retroviruses. Adenoviral-mediated gene transfer allows for high transient overexpression of the transduced gene.
The improvement of adenoviral vector design has to deal with the problem of immunogenicity. Most likely, the leaky E2 gene expression of the adenoviral vector system is responsible for the toxicity and inflammatory reactions. Studies are currently in progress to design new generations of adenoviral vectors lacking E2a-gene functions, either by mutations [110, 111] or by deletion of E4 genes, which requires the construction of helper cell lines that can provide E4-function [112, 113].
Other strategies that are currently pursued to avoid immune responses are directed at reducing viral load by developing high-efficiency transgene expression vectors in combination with short-term immune suppression [114, 115] and/or by generating chimeric adenoviruses type 5 carrying fiber genes of adenovirus type 7 [116]. The advantage of using such a chimeric capsid is the binding affinity enhancement of the adenoviral particle to the target cell.
Adenoviral/Retroviral Chimeric
Vectors
A chimeric adenoviral/retroviral vector system has
recently been developed [66] in order to combine the advantages of
adenoviruses and those of retroviruses in a single gene transfer
system. This may allow for the simultaneous achievement of more
efficient gene delivery and longer-term transgene expression. Both
features are necessary to optimize the in vivo therapeutic gene
transfer interventions to correct human defective genes. Briefly, this
gene delivery system consists of an adenoviral vector carrying in its
genome the packaging components of a retrovirus together with the
retroviral transfer vector, which is the recipient for transgenes. As
already mentioned, the adenoviral vector can be produced at very high
titers and can also infect nondividing cells. The adenoviral genome is
transiently overexpressed in transduced cells, as it is not integrated
into the host genome. At this stage, the transduced cells produce
retroviral vectors capable of infecting other surrounding cells. This
may improve the efficiency of in vivo retroviral transduction. Once
certain tissues have been infected by the chimeric
adenoviral/retroviral vector system, retroviral vectors are produced
in vivo over a considerable period of time and can reach their target
cells. The constitutive localized production of retroviral vectors
may, at least partially, overcome the complex issue of
complement-mediated lysis of retroviral particles that occurs in the
in vivo model. However, this system needs to be improved and better
characterized before it can be applied in clinical trials; the
immunogenicity of adenoviral vectors must be completely
devoided; there is still the possibility of proviral
insertional cell mutagenesis; the retroviral titers are still too low
for effective in vivo applications.
AAV-Based
Vectors
AAV is a human parvovirus that does not seem to be
associated with any human disease [27]; therefore, the first requirement for gene
therapy applications is easily accomplished. In addition, AAV has many
desirable properties: it can infect a wide range of cells deriving
from different tissues [28]; it can also infect nondividing cells
[30, 117]; it can establish a latent
infection by integrating its genome [29]; the integration of the viral genome is
site-specific for the q arm of chromosome 19, between q13.3 and qter
[31-35]. All of these properties
explain the considerable interest in applying AAV as a vector in gene
therapy. The site-specific integration of AAV is a desired safety
feature that is, however, lost in AAV recombinant vectors. The major
research aim is to conserve the site-specific integration of AAV
vector systems, possibly by cotransfecting a plasmid encoding the
protein Rep78, which seems to be responsible for the viral-specific
integration process in the presence of the inverted terminal repeats
[118, 119]. Other problems for the
application of AAV-based vector systems are related to the limited
capacity of accommodating foreign genes, that is, those in the range
of 4.1-4.9 kb [120]; to
the difficulty of obtaining pure high-viral titers, and the
requirement for helper adeno- or herpesvirus for replication in cell
culture [121-123]. The inability to completely
eliminate helper viruses has raised an element of concern about the
application of AAV vectors in clinical trials.
In preliminary experiments, recombinant AAV vectors have stably transduced a certain number of nondividing cells, such as hematopoietic progenitor cells [124], neurons [125], and photoreceptor cells [126]. Another encouraging finding is the lack of immune response to in vivo AAV-mediated-gene transfer [127]. It is likely that recombinant AAV vectors will be employed for the treatment of cystic fibrosis [128] instead of adenoviruses.
Cationic Liposomes and Other
Nonviral Vector Systems
Nonviral vector systems comprise
various formulations of cationic liposomes [129-131] and composite vectors devised for gene
delivery applications by receptor-mediated entry containing a
DNA-binding moiety, a receptor-targeting molecule, and often a
lysosome-breaking agent [132-135].
These gene delivery systems are not infectious and have a low toxicity. Theoretically, there is no limit to the DNA size that liposome particles can carry. Furthermore, liposome-based vector systems are suitable for the delivery of oligonucleotides to mammalian cells. Receptor-mediated gene delivery systems have the additional advantage of a potentially specific target. The disadvantages of both systems are low transfection efficiency and the transiency of gene expression. Cationic liposomes have the additional disadvantage of lack of specific targeting, whereas receptor-mediated delivery systems may be immunogenic.
Cationic liposomes have already been employed in phase I clinical trials for the treatment of cystic fibrosis [136].
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Conclusion |
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TopAbstractIntroductionGene Transfer
ModelsVector Design for In...ConclusionReferences |
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The standpoint of gene therapy basic research is still far from providing the tools for the treatment of the previously mentioned illnesses. The most pressing issue that the field of gene therapy has to address is the development of efficient in vivo gene delivery systems. The in vivo administration of either functional genes or therapeutic factors would greatly simplify and improve any human gene therapy intervention.
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ACKNOWLEDGMENT |
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TopAbstractIntroductionGene Transfer
ModelsVector Design for In...ConclusionReferences |
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Reprinted from The Oncologist 1998;3:225-236.
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TopAbstractIntroductionGene Transfer
ModelsVector Design for In...ConclusionReferences |
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