Review Virus-mediated gene delivery for human gene therapy · Review Virus-mediated gene delivery...

Review

Virus-mediated gene delivery for human gene therapy

Mauro Giacca ⁎, Serena ZacchignaMolecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy

a b s t r a c ta r t i c l e i n f o

Article history:Received 27 October 2011Accepted 3 April 2012Available online 10 April 2012

Keywords:AdenovirusAdeno-associated virusGene therapyRetrovirusViral vectors

After over 20 years from the first application of gene transfer in humans, gene therapy is now a mature dis-cipline, which has progressively overcome several of the hurdles that prevented clinical success in the earlystages of application. So far, the vast majority of gene therapy clinical trials have exploited viral vectors asvery efficient nucleic acid delivery vehicles both in vivo and ex vivo. Here we summarize the current statusof viral gene transfer for clinical applications, with special emphasis on the molecular properties of themajor classes of viral vectors and the information so far obtained from gene therapy clinical trials.

© 2012 Elsevier B.V. All rights reserved.

Contents

1. Genes as medicines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3772. Viral vectors: the perfect biological nanoparticles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378

2.1. Gammaretroviral vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3802.2. Lentiviral vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3812.3. Adenoviral vectors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3812.4. Vectors based on the herpes simplex virus type 1 (HSV-1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3822.5. Vectors based on the adeno-associated virus (AAV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382

3. Lessons on gene therapy vectors learned from preclinical experimentation and clinical trials . . . . . . . . . . . . . . . . . . . . . 3834. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386

1. Genes as medicines

The concept to use genes as drugs for human therapy, originallyconceived around 1970, was the logical consequence of at least twomajor advances occurring at the time, namely the exponential growthin the knowledge of human gene function and the impact of their mu-tations, and the progressive development of more effective technolo-gies for DNA delivery into mammalian cells [1]. Initially, gene therapywas synonymous for supplying a missing cellular function by trans-ferring a normal copy of an otherwise mutated gene into the relevanttarget cells. This concept applies little to the current advancement ofgene therapy. While replacement gene therapy is obviously at the

basis of the vast majority of gene therapy clinical trials for inheriteddisorders, these represent less than 8% of all clinical trials so far con-ducted. In most other instances, protein-coding cDNAs are used tomodulate cell behavior (Table 1).

Key examples of this application include, among others, the blockof cancer cell proliferation by interfering with cell cycle regulatoryproteins; immune cell activation by transferring genes coding forco-stimulatory proteins into cancer cells; the secretion of growth fac-tors and cytokines coding for neurotrophic factors in Parkinson's orAlzheimer's diseases, and the production of angiogenic factors in pe-ripheral or cardiac ischemia. For an extensive review of these applica-tions, cf. ref.: [2].

Even more notably, besides protein-coding nucleic acids, the spec-trum of gene therapy applications is enormously increased by thepossibility to use small nucleic acids (DNAs or RNAs) with regulatoryfunction. These molecules now belong to one of at least six possible

Journal of Controlled Release 161 (2012) 377–388

⁎ Corresponding author at: ICGEB Trieste, Molecular Medicine Laboratory, Padriciano,99, 34149 Trieste, Italy. Tel.: +39 040 375 7324; fax: +39 040 375 7380.

E-mail address: [email protected] (M. Giacca).

0168-3659/$ – see front matter © 2012 Elsevier B.V. All rights reserved.doi:10.1016/j.jconrel.2012.04.008

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classes, namely DNA oligonucleotides, small catalytic RNAs and DNAs(ribozymes and DNAzymes respectively), small regulatory RNAs(siRNAs and microRNAs), long antisense RNAs, decoy RNAs and DNAsand RNAs binding to other molecules thanks to their tridimensionalstructure (aptamers) (Table 1). Notably, DNA oligonucleotides mustbe administered to the cells from the outside. In contrast, all RNA thera-peutics, similar to protein-coding cDNAs, can also be synthesized insidethe cells by transferring their coding DNA sequences.

2. Viral vectors: the perfect biological nanoparticles

In most instances, the efficiency of gene transfer continues to repre-sent the most relevant obstacle limiting the clinical success of genetherapy. Given the broad spectrum of characteristics and mechanismsof action displayed by both coding and non-coding nucleic acids, it isevident that no perfect universal system for their delivery exists. In allcases, however, the apolar and hydrophobicmembranes of mammalian

Table 1Spectrum of therapeutic nucleic acids used by gene therapy and examples of their clinical applications. For an extensive description, cf. ref. [2].

Category Type of nucleic acid Examples of clinical application

Protein-codingDNA sequences

Proteins substituting missing or mutated cellular proteins Replacement therapy for Duchenne muscular dystrophy, lysosomal storage disorders,hemophilia and several other inherited disorders

Proteins modulating cellular functions Costimulatory proteins (e.g. B7, ICAM-1, LFA-3) to activate cytotoxic T lymphocytes; HIV-1RevM10 mutant to block HIV-1 replication

Secreted growth factors and cytokines Neurotrophic factors in Parkinson's and Alzheimer's diseases; VEGF in myocardial and pe-ripheral ischemia

Proteins regulating cell survival and apoptosis HSV-1 thymidine kinase prodrug gene therapy to induce cell death in neuroblastoma; Bcl-2for amyotrophic lateral sclerosis

Antigens for vaccination Antitumor and antiviral vaccination

Antibodies and intracellular antibodies Intracellular antibodies against HIV-1 integrase, Rev or reverse transcriptase to block viralreplication; intracellular antibodies against antiapoptotic proteins in cancer

T-cell receptor (TCR) subunits Modified T-cell receptor subunit genes to retarget immune response towards tumor andviral antigens

Non-codingnucleic acids

Oligonucleotides andmodifiedoligonucleotides

Phosphorothioate oligonucleotides Oligonucleotides blocking gene expression (e.g. to inhibit viral replication or to inhibitexpression of a proapoptotic protein in cancer); oligonucleotides modulating pre-mRNAsplicing (e.g. to induce exon skipping in Duchenne muscular dystrophy)

2′-Ribose modified oligonucleotidesLocked nucleic acids (LNA) andethylene-bridged nucleic acids(ENA)Morpholinos (PMO)Peptide nucleic acids (PNA)

Catalytic RNAs and DNAs Ribozymes and DNAzymes Targeting pathological alleles in dominant inherited disorders; targeting viral mRNAs toinhibit viral infectionSmall regulatory RNAs siRNAs and shRNAs

MicroRNAs Modulating cell function (e.g. stimulation of myocardial cell proliferation after myocardialinfarction)

Long antisense RNAs Inhibition of viral gene expression (e.g. inhibition of HIV-1 replication)

Decoys Sequestering a factor essential for viral replication (e.g. Rev protein in the course of HIV-1infection)

Aptamers Sequestering a relevant growth factor (e.g., VEGF in the treatment of age-related maculardegeneration)

Fig. 1. Schematic representation of the organization of the viral genomes of gammaretrovirus, lentivirus (HIV-1), AAV and adenovirus (left side), and of the corresponding vectorsgenerated from these viruses (right side). Gammaretrovirus and HIV-1: the common retroviral genes (gag, pol and env) are shown in dark green; the HIV-1 accessory genes in lightergreen; the long terminal repeats (LTRs) are boxed; the localization of genetic elements relevant for vector production (primer binding site, PBS, packaging signal, ψ, 5′ and 3′ splicesites, 5′ and 3′ ss, polypurine tract, PPT, central poly-purine tract, cPPT and Rev-responsive element, RRE) is indicated; on the vector side, the localization of the therapeutic gene isshown in orange and that of the remaining portions of the viral genes (gag, pol, env) with the respective initial letters (g, e, p) boxed in green; RSV: Rous Sarcoma Virus promoter,used to express the retroviral mRNA in the packaging cells; RU5: portions of the LTR left intact in SIN vectors. AAV: the viral genes (rep and cap) are shown in light green; the lo-calization of promoter is shown by arrow; ITR: inverted terminal repeat; poly A: polyadenylation site on the vector side; P: promoter. Adenovirus: viral genes present in the both thewt virus and the vectors are shown in dark blue, with arrows indicating the direction of transcription; the genes removed in first generation adenoviral vectors (E1A, E1B, E3) areshown in light blue; the localization of the therapeutic gene and its promoter (P) is shown in orange; ITR: inverted terminal repeat; ψ: packaging signal. Herpes simplex virus 1: TheHSV-1 genome consists of a linear, double-stranded DNA molecules of 152 kb containing more than 80 genes. The genome is composed of unique long (UL) and unique short (US)segments which are flanked by inverted repeats. These are designated as TRL and IRL (terminal and internal repeat of the long segment, respectively) and TRS and IRS (terminal andinternal repeat of the short segment). The repeats surrounding UL are designated ab and b′a′, while those surrounding US are designated a′c′ and ca. There are two different originsof replication, oriL in the long segment and oriS in the short segment. OriS is duplicated, along with ICP4, because they are found in the inverted repeats surrounding the long seg-ment. Approximately half of the genes are essential for viral replication in cell culture (listed on top); the other half are non essential for viral replication in cultured cells (bottom).Genes in blue are non-essential genes that are mutated in the replication-competent (attenuated) viruses so far developed and described in the text; genes in red are immediateearly (IE) genes that are mutated in the replication-defective viruses. The genome contains three pac signals (shown in yellow) that assist in packaging the viral genome DNA intovirions.

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cells, at either the plasma membrane or endosomal levels, represent aformidable barrier for any large polyanion such as DNA or RNA. Withvery few exceptions, naked nucleic acids are, therefore, very poorlyinternalized by the cells when administered in the extracellular milieu.Gene transfer must be facilitated using physical (for example, electro-poration or high pressure injection), chemical (cationic lipids orpolymers) or biological (viral vectors) tools.

In light of the several hundred clinical trials conducted so far— seebelow, it is immediately apparent that the vast majority of experi-mentations have exploited genetic modification of viruses to transfernucleic acids into the target cells. This is not surprising since, in theirreplicative cycle, viruses make use of very efficient mechanisms to inter-nalize their own genome into the target cells, which have evolved overmillions of years. In the most simplistic view, a viral particle is a

379M. Giacca, S. Zacchigna / Journal of Controlled Release 161 (2012) 377–388

nanoscale-sized object composed of a nucleic acid and a few proteins thatimpede its degradation in the extracellular environment and mediate itsinternalization into the target cells. A perfect viral vector, therefore, ex-ploits the biological properties of such a particle by substitutingmost ofthe viral genome with the therapeutic nucleic acids of interest.

When considering the characteristics of all viral vectors availablefor gene therapy, it becomes apparent that the principles accordingto which these are obtained are common to all systems. They consistin: i) the removal of most genes coding for viral proteins from theviral genome, and, in particular, of those that are potentially pathogenic;ii) maintenance of the cis-acting sequences of the viral genomes re-quired for viral replication; in particular, those determining inclusionof the genomes within the viral particles (packaging signal, ψ); iii) ex-pression of the viral proteins required for viral replication within thevirus-producing cells (called packaging cells); these proteins can beexpressed from genes encoded by transiently transfected plasmids orby a previously engineered cellular genome, or by a helper virus simul-taneously infecting the packaging cells.

Here we focus on five classes of viruses, which are those utilized inmore than 50% of human gene therapy clinical trials; these includemembers of the Retroviridae family (gammaretroviruses and lentivi-ruses), adenoviruses, adeno-associated viruses (AAVs) and herpesvi-ruses. In the following sections, we summarize the most relevantmolecular properties of these viruses with respect to their utilizationas gene therapy vectors. A comparative analysis of the genetic organi-zation of these viruses and that of the corresponding vectors is shownschematically in Fig. 1.

2.1. Gammaretroviral vectors

Viral vectors based on gammaretroviruses have been the most uti-lized in gene therapy clinical trials until the early 2000s. Their initialpopularity, due also to their relative simplicity of use, high efficiencyof transduction of replicating cells (e.g. ex vivo cultured cells), lowimmunogenicity and property to integrate their proviral cDNA forminto the host cell genome, with the potential to render transduction,and thus therapeutic gene expression, permanent [3].

The genomes of prototypic members of the gammaretrovirusgenus of the Retroviridae family, such as the Moloney-murine leuke-mia virus (Mo-MLV), are 9–11 kb long and consist of 3 essentialgenes (gag, pol and env) flanked, in their integrated, proviral DNAforms, by two identical sequences of 400–700 bp at the 3′ and 5′ ex-tremities, the long terminal repeats, LTRs (Fig. 1). Besides these genes,at least 5 genetic elements are necessary for the completion of theviral replicative cycle and thus essential for the construction of vec-tors. These are (from the 5′ to the 3′ of the genome): the LTRs, ofwhich the 5′ U3 region is the promoter for mRNA transcription, theR region is required for reverse transcription and the 3′ U5 regioncontains the polyadenylation site; the primer binding site (PBS), posi-tioned immediately downstream of the 5′ LTR, which is required forcellular tRNA binding to prime reverse transcription; the 5′ and 3′splice sites (5′ and 3′ ss respectively), which are essential to generatethe spliced mRNA used for the translation of env; the packaging signal(ψ), which includes a structured RNA region at the 5′ of the gag gene,partially extending toward the 5′ ss; this is the sequence binding toGag that is required for the inclusion of the viral genomemRNA insidethe virions during assembly; the polypurine tract (PPT), positioned atthe 3′ end of the genome upstream of the 3′ LTR, which is required forreverse transcription [3].

Gammaretroviral vectors must contain these five genetic ele-ments, while the rest of the genome is dispensable and can be re-moved and substituted by the therapeutic gene, including thesequences coding for the viral proteins (Fig. 1). Thus, in the simplestversion of these vectors, transcription of the therapeutic gene is di-rectly controlled by the viral 5′ LTR.

Viral vectors based on gammaretroviruses are produced in cul-tured mammalian cells. In the early days of gene therapy, a plasmidcontaining the proviral DNA was transfected into a packaging cellline, usually of murine origin, that expressed the retroviral gag, poland env genes, no longer present in the retroviral vector plasmid[4,5]. Once transfected into such a packaging cell, the plasmid con-taining the retroviral vector was transcribed starting from the 5′LTR and thus generated an mRNA encompassing the whole proviralconstruct and containing the packaging signal (ψ). Presence of thissignal permitted recognition of the vector mRNA by Gag, followedby its inclusion into a fully infectious virion displaying the env geneproduct on its surface. A gammaretroviral virion generated in thismanner is indistinguishable from a wild type virion, and is thusfully infectious. After infection of a target cell, via reverse transcrip-tase (present inside the virion) and the cis-acting PBS and PPT se-quences, the vector genome is reverse transcribed. The proviralcDNA is then integrated into the host cell genome by integrase,which is also present in the virion. Once integrated, the mRNAexpressed by the vector provirus is no longer infectious, since noneof the retroviral proteins are present. Thus, retroviral vectors areonly capable of a single cycle of infection.

Since tropism and transduction efficiency of retroviral vector parti-cles depend on the interaction of the env gene product with cellularreceptors, in more recent years, it has became evident that thismight significantly increase by pseudotyping the virions through thesubstitution of env proteins with other membrane proteins that aremore efficient at driving the fusion process between the virion andthe target cells and at expanding tropism. A very effective protein forthese purposes is the G protein of the vesicular stomatitis virus(VSV), an enveloped virus with a negative sense RNA genome belong-ing to the family of Rhabdoviridae. VSV-G mediates infection by bind-ing the phospholipids present on virtually all mammalian cellmembranes at very high efficiency and triggering endocytosis of theviral particles [6]. Once in the endocytic compartment, the loweringof the pH activates the fusogenic properties of VSV-G, which deter-mines fusion of the viral envelope with the endosomal membraneand release of the virion content into the cytosol. Due to its fusogenicproperties, it is nonetheless, not possible to permanently express VSV-G in a packaging cell line. VSV-G-pseudotyped retroviral vectors arethus obtained by the transient transfection of packaging cell linesthat only express Gag–Pol with a plasmid expressing VSV-G underthe control of a strong promoter (such as the promoter of the cyto-megalovirus immediate-early genes), in addition to the plasmid con-taining the retroviral vector DNA [7]. In contrast to virionscontaining retroviral Env proteins, VSV-G-pseudotyped virions canbe purified by high speed centrifugation without significant loss of in-fectivity. By using a single packaging passage followed by centrifuga-tion, titers to the order of ~1×108–1×109 infectious particles/ml ofsupernatant can routinely be obtained.

VSV-G-pseudotyped retroviral vectors have broad species speci-ficity and cell type range, but have their disadvantages as well. Forcertain, in vivo gene-therapy applications, it may be important to re-strict the transfer and expression of the gene to specific cell types. Fol-lowing systemic administration, the VSV-G pseudotyped vectorstransduce target cells of interest, but can also transduce other celltypes that would have undesirable effects in gene-therapy protocols.For example, antigen-presenting cells (APCs) could cause deleteriousimmune responses if they are transduced inadvertently. Unfortunate-ly, VSV-G-pseudotyped lentivectors can efficiently transduce APCsfrom mice and humans, and can elicit immune responses againstthe transgene product [8], thus potentially negating the therapeuticeffects of the protein expression.

In more recent years, other pseudotyping proteins have been in-troduced in the gene therapy arena, mainly with the purpose of eitherenhancing or rendering more specific the tropism from a specific celltype; in most instances, these proteins can be used to pseudotype


both gammaretroviral and lentiviral vectors (cf. below). For instance,efficient local and dispersed neuronal transduction in the central ner-vous system can be obtained using RVG/VSVG chimeric proteins, con-sisting in a fusion product between the external domain of Rabiesvirus glycoprotein (RVG) and the cytoplasmic domain of VSV-G[9,10]. In contrast, the use of the non-neurotropic envelope glycopro-tein of the lymphocytic choriomeningitis virus (LCMV-GP) allowsspecific infection of glioma cells while sparing neurons, thereby wid-ening the therapeutic window of retroviral vectors from cancer celltherapy in the brain [11].

Other attempts have been made to enhance targeting of dendriticcells, with the purpose to deliver effective anti-tumor immunity forcancer immunotherapy. The most promising approaches validated sofar include the use of an engineered viral glycoprotein derived fromthe Sindbis virus [12], the major envelope protein of baculovirus,gp64 [12], or a few envelope glycoproteins derived from the Auravirus (AURA), a member of the alphaviruses genus, which increasesthe ability of retroviral vectors to transduce dendritic cells by bindingto C-type lectins, widely exposed on the surface of myeloid cells [13].

2.2. Lentiviral vectors

One of the most striking characteristics distinguishing viruses ofthe Lentivirus genus (prototype: HIV-1) from gammaretroviruses istheir ability to infect non-replicating cells. This is due to the capacityof the lentiviral pre-integration complex (PIC), which forms in the cy-tosol, to actively cross the nuclear membrane thanks to the interac-tion of some of the PIC proteins (integrase, matrix, Vpr) withproteins of the nuclear pore [14]. This property is of paramount inter-est for gene therapy, since it allows a significant extension of therange of cell types in which gene transfer might be of therapeuticbenefit, especially because most of the cells in our body are non-replicating. In addition, in the case of hematopoietic stem cells, exvivo transduction with lentiviral vectors appears efficient also in theabsence of growth factor stimulation, a condition permitting the pres-ervation of their pluripotency [15]. Mainly for these reasons, since thelate 1990s, the possibility to obtain vectors based on HIV-1 and otherlentiviruses has appeared very appealing for in vivo and ex vivo genetransfer applications [15,16].

In addition to gag, pol and env, HIV-1 also contains an extra series ofsix, so-called “accessory genes” (tat, rev, nef, vpr, vpu and vif), whichare nevertheless essential for different steps of the viral life cycle, in-cluding transcription (tat), transport of viral mRNAs outside of the nu-cleus (rev), cell cycle regulation (vpr) and modulation of virioninfectivity (vif); Fig. 1. Since 1996 [17], at least three generations oflentiviral vectors have been produced, by progressively omittingmost of these genes from the vector production system, with the ulti-mate goal of ensuring safety [18]. The third and current generation ofHIV-1-based, lentiviral vectors only requires 3 of the 9 HIV-1 genes,thus offering a safety profile that is definitely reassuring [19]; Fig. 1.Production of these vectors, which are now in clinical experimenta-tion, requires four plasmids. The first plasmid corresponds to the ther-apeutic gene transfer vector, in which the 3′ U3 LTR region is deleted,to inactivate transcription of the proviral DNA after reverse transcrip-tion (self-inactivating— SIN— vector [20]). In the packaging cells, thevector is transcribed from a constitutively active heterologous pro-moter, positioned upstream in the LTR R region. In this construct, thepol gene retains a sequence, named the central polypurine tract/centraltermination sequence (cPPT/CTS) that increases viral titers by enhancingboth reverse transcription and PIC nuclear transport [21,22]. A secondplasmid (packaging plasmid) contains the gag and pol genes while athird plasmid the rev gene, which is required, allows proper transportinto the cytosol of themRNA expressed from the packaging plasmid. Fi-nally, a fourth plasmid encodes VSV-G. Infectious viral particles areobtained by transiently transfecting human embryonic kidney 293 T(HEK 293T, expressing the SV40 T antigen protein) cells with these

plasmids; infectious virions are then found in the cell culture superna-tant, similar to gammaretroviral vectors.

2.3. Adenoviral vectors

Currently, over 100 members of the Adenoviridae family, able toinfect man and a wide number of different animal species, areknown; human adenoviruses are responsible for 5–10% of acute re-spiratory diseases in children and a variable number of epidemic con-junctivitis and gastroenteritis.

The natural tropism of adenoviruses for the respiratory epitheliumand the conjunctiva is mainly due to its mode of transmission ratherthan to the molecular characteristics of the virus. Indeed, the receptormediating cell infection by adenoviruses (the coxsackie/adenovirusreceptor, CAR) is ubiquitously expressed, and most of the cell typescan sustain adenoviral replication independent from the replicativestate of the cells [23]. An additional attractive property of adenovi-ruses is the great efficiency with which they exploit the cellular ma-chinery to drive synthesis of viral mRNAs and translation of viralproteins. Given these considerations, it is not surprising that, sincethe second half of the 1990s, adenoviral vectors have become thefocus of a vast series of both animal and clinical experimentations.Based on the capacity of different human sera to neutralize adenoviralinfection in cell culture, more than 50 serotypes capable of infectinghumans can be distinguished; these are then classified into 6 sub-groups (A–F) on the basis of their capacity to determine human redblood cell agglutination. Most of the gene therapy vectors derivefrom serotypes 2 and 5 (Ad2 and Ad5) of subgroup C.

The adenoviral genome consists of a double-stranded, linear DNAmolecular of 36 kb in the case of Ad2 and Ad5, bearing two identicalsequences in reverse orientation at the two extremities (inverted ter-minal repeats, ITRs; 103 bp in the case of Ad2 and Ad5). The genomecontains five early transcriptional units, which become activatedupon cell infection (E1A, E1B, E2, E3 and E4), two delayed early tran-scriptional units (IX and IVa2) and one major late (ML) transcriptionunit, which is processed to generate 5 families of late mRNAs throughpost-translational processing (from L1 to L5); Fig. 1.

A first generation adenoviral vector is obtained by substituting theE1, or the E1 and E3, regions with an expression cassette, consisting ofthe therapeutic gene, a promoter and a polyadenylation site [24].Since E1 proteins are required for viral replication to produce the vec-tor particles, these are supplied in trans by specific cell lines, such asHEK 293, 911, N52.E6 or PER.C6 [24]. The E3 region codes for proteinsthat are important to counteract the host antiviral mechanisms, how-ever are not required for in vitro adenovirus replication, and are thusnot complemented for vector production. Vectors carrying deletionsin only E1 can accommodate foreign DNA stretches of up to 5.1 kb,while those deleted in E1 and E3 can do so up to 8.3 kb [25]; Fig. 1.

While the E1-deleted vectors cannot replicate in vivo, expressionof the several adenoviral genes that are still present stimulates a pow-erful inflammatory response of the host, which raises important safe-ty concerns, as will be further discussed below. In addition, theimmune response limits the duration of therapeutic gene expressiondriven by these vectors, since the transduced cells are eliminated bycytotoxic T lymphocytes [26–28]. To overcome these problems, a sec-ond generation of vectors was obtained, bearing additional deletionsin the E2 and E4 regions. These vectors could accommodate up to14 kb of foreign DNA [29]. Despite the elimination of these genetic re-gions, these vectors did not completely solve the issue of adenovirus-induced toxicity, given the immunogenic and inflammatory potentialof the residual genes. Furthermore, expression of the therapeuticgene from these vectors was reduced compared to first generationvectors, probably because some of the E2 and E4 genes code for pro-teins that directly or indirectly increase the levels of expression of thevirus-encoded genes.


Finally, a third generation of adenoviral vectors is characterized bythe complete deletion of the adenoviral genome and its substitutionwith exogenous DNA, with the exception of the regions required incis for viral DNA replication and packaging (ITRs and ψ respectively).These vectors are named gutless or gutted or, more appropriately,helper-dependent (since their replication depends entirely on co-infection of the cells in which packaging occurs with a helper vectorproducing, in trans, all the required proteins) or high-capacity (HC,since they can accommodate up to 37 kb of exogenous DNA, thus alsoallowing delivery of large DNA sequences or multiple genes) [30,31].

Production of adenoviral vectors requires a two-step procedureentailing, first the generation of a vector genomic DNA with the se-quence of interest and later, its replication and packaging to obtaininfectious viral preparations. The production of first and second gen-eration vectors is essentially based on the generation of long mole-cules of linear DNA corresponding to the desired adenoviral vectorgenome by recombination, which can occur in helper cells, bacteriaor in vitro [24]. Alternatively, gutless vectors, which are devoid ofviral genes except for the ITRs at the two extremities and the ψ regioninside, are obtained by providing all the proteins necessary for vectorDNA replication in trans using a replication-competent adenovirus,acting as a helper [32].

2.4. Vectors based on the herpes simplex virus type 1 (HSV-1)

HSV-1 has broad host range, high natural infectivity of both repli-cating and non-replicating cells and capacity to establish a latent in-fection in neurons. Since the early days of gene therapy, all theseproperties have appeared very appealing in view of developing viralvectors. However, the relative complexity of the viral genome andour still incomplete knowledge of the molecular properties of variousviral proteins still hamper a wider utilization of this vector system.

Three different kinds of vectors derived from HSV-1 are currentlyconsidered: attenuated vectors, replication-defective vectors andamplicon vectors.

Attenuated vectors are still capable to replicate but exhibit attenu-ated virulence due to the removal of genes dispensable for in vitro butessential for in vivo replication, such as the enzyme thymidine kinase(ICP36/UL23), or the major subunit of ribonucleotide reductase(ICP6/UL39), which blocks mRNA translation in the infected cells, orthe neurovirulence factor ICP34.5 [33]. Several studies have shownthat these vector types not only replicate once inoculated into thebrain, but also diffuse to distant areas [34]. The major application ofthese vectors is for the oncolytic therapy of cancer, either alone orin combination with chemotherapy [35,36]. The newest generationof these vectors, in addition to deletions in the above mentionedgenes, also contains genes coding for various cytokines (IL-4, IL-12,IL-10, GM-CSF) or for the co-stimulatory molecule B7.1, with the ulti-mate purpose to increase tumor immunogenicity [37–40].

Within the class of replication-defective vectors, a first generationwas obtained consisting of mutants deleted in the single essentialimmediate-early (IE) gene encoding ICP4 [41]. Since these vectorsshowed reduced pathogenicity in the brain but still retained neuro-toxicity in cell culture, they were further improved by the introduc-tion of deletions in additional genes [42]. These multiply-deletedviruses show prolonged persistence in vivo and offer the possibilityof cloning multiple therapeutic gene cassettes in different regions oftheir genome [37].

Finally, amplicon vectors are viral particles identical to wild typeHSV-1 virions, in which the genome consists of concatameric copiesof the amplicon, namely a plasmid containing the ori-S origin and apackaging signal (pac) derived from the HSV-1 genome, in additionto a therapeutic gene cassette [43]. Since HSV-1 virions can packageDNA molecules that can extend to over 150 kb, herpesviral ampliconsare the viral vector system offering the largest cloning capacity cur-rently available. The production of amplicon vectors is obtained by

co-transfecting the amplicon with a set of 5 partially overlapping cos-mids, expressing all the required viral proteins, or a single bacterialartificial chromosome (BAC), as source of the viral proteins requiredfor particle production. Both replication-defective and amplicon vec-tors are packaged into complete HSV-1 particles to infect targetcells; however, amplicons persist inside the infected cells without ex-pressing any viral protein, thus avoiding any possible problem ofreactivation and virulence.

The replication-defective and amplicon vectors have been used atthe preclinical level to express a variety of genes in both the nervoussystem (for example, neurotrophic factors for gene therapy of neuro-degenerative disorders [44]) and other tissues such as muscle, heart,liver [45–47], or for genetic vaccination [48].

2.5. Vectors based on the adeno-associated virus (AAV)

AAV is a member of the Dependovirus genus of the Parvoviridaefamily (parvo-: Latin for “small”), which includes a vast series ofsmall viruses with icosahedral symmetry, without envelope, contain-ing a single-stranded DNA genome, infecting numerous species ofmammals, including man [49]. In primates alone, over 100 AAV vari-ants have been discovered to date, and new serotypes are continu-ously identified (i.e. variants with different antigenic properties, notrecognized by the currently available antisera). More than 80% ofadults 20 years of age or older show an antibody response againstAAV, proving that they have encountered the virus, probably intheir infancy [50]. Despite their diffusion, no dependoviruses haveever been associated with any human disease.

AAV virions are the smallest among gene therapy vectors. Theyhave a capsid with icosahedral symmetry with a diameter of18–25 nm, composed of only 60 proteins encoded by a single gene(the cap gene). The capsid includes the viral genome, consisting of alinear single stranded DNA, with either positive or negative polarity;in any AAV preparation, about half of the virions have a DNA withpositive polarity, the rest have a DNA with negative polarity [51].

In recent years, at least 12 different AAV serotypes have been iso-lated (AAV1–AAV12) and well characterized antigenically [52,53]. Allthese viruses share similar structure, size and genetic organizationand only significantly differ in the amino acid composition of the cap-sid proteins, which dictates receptor specificity. All AAVs use recep-tors that are ubiquitously and abundantly expressed. The serotypemost utilized so far, both experimentally and clinically, is AAV2,which binds to cell surface heparan sulfate proteoglycans (HSPGs);αvβ5 integrin and the receptors for fibroblast growth factor (FGFR-1)and hepatocyte growth factor (HGFR) function as co-receptors insome cells [12,29,54,55]. Similar to AAV2, AAV3 also binds HSPGs [56].In contrast, AAV1, AAV4, AAV5 and AAV6 interact with sialic acid(N-acetylneuraminic acid, Neu5Ac) residues, linked with variousbonds to the cell surface glycans [56–59]. AAV8 binds a specific cell sur-face protein, LamR, which exerts several functions in the cells, includingthat of receptor for extracellular laminin [60]. AAV2 and AAV5 particlesenter cultured cells by clathrin-mediated endocytosis and are found inearly endosomes immediately after entry [61,62]. These cellular com-partments traffic through the cytoplasm and rapidly approach a peri-nuclear location, where they mature into late endosomes [63].

The use of capsids with serotypes that are different from AAV2 onone hand increases efficiency of transduction in the already permis-sive cell types while, on the other, extends tropism to a few other or-gans. For example, the skeletal muscle is transduced with particularefficiency by AAV1 and AAV6 (which differs from AAV1 in only 6amino acids) [64]; in the retina, photoreceptors are an efficient targetof AAV5, AAV7 and AAV8 while the pigment epithelium of AAV5 andAAV4 [65]; finally, AAV8 transduces both the endocrine and exocrinepancreas in addition to the liver [66,67]. None of the serotypes, how-ever, permit significant transduction of cells physiologically refractory


to AAV2 gene transfer, amongwhich endothelial cells, fibroblasts, var-ious types of stem cells and others.

Another very interesting property of some of the most recent AAVserotypes, AAV8 and AAV9, is their capacity to cross the endothelialbarrier of blood vessels. Once injected intravenously or intraperitone-ally in the experimental animal, these vectors reach the skeletal mus-cle parenchyma and transduce myofibers at high efficiency [68]. Thus,they represent potential tools for whole muscle transduction for genetherapy of muscle dystrophies [69].

A molecular explanation for the different cell-type selectivity ofthe various AAV serotypes is still missing. It is conceivable that theuse of different entry molecules route the vectors towards differentpathways, or that the various capsid proteins modulate the interac-tion of the viral genome with cellular proteins differently.

The single-stranded AAV genome has about 4.7 kb and containstwo open reading frames, corresponding to two genes, rep and cap(Fig. 1), which code for the proteins necessary for viral replicationand for forming the viral capsid, respectively. These coding regionsare flanked by two ~145 nt long inverted terminal repeats (ITRs),with an internal complementarity stretch in their first 125 nt andthus forming a T-shaped hairpin structure, identical at the two viralends. This palindromic sequence is the only cis-acting genetic ele-ment necessary for all AAV functions, including viral DNA replication,site-specific integration into the host cell DNA and packaging of vi-rions. The first two activities (replication and integration) requirethe presence of Rep68 or Rep78 proteins, which specifically bind a se-quence within the ITR, the Rep binding site (RBS), and cleave in a site-and strand-specific manner at the terminal resolution site (TRS) lo-cated 13 nucleotides (nt) upstream of the RBS [70]. An almostidentical sequence in human chromosome 19q13.4 (AAVS1, locatedin a centromeric position with respect to the genes coding for theslow skeletal muscle troponin T (TNNT1) and cardiac troponin I(TNNI3) [71,72]) represents the minimal sequence necessary and suf-ficient for AAV site-specific integration. The two ITRs are the only AAVsequences preserved in the vectors, while a transcriptional cassette(promoter+therapeutic gene+polyadenylation site) substitutesthe rest of the genome. Interestingly, since Rep is not expressed bythe vectors, the viral genome does not integrate in the host cell andremains episomal forming concatamers (reviewed in refs.: [73,74]).

AAV vectors are usually obtained from the AAV2 genome andcloned in a plasmid form by removing all the viral sequences withthe exception of the two ITRs (about 145 bp each). Between theITRs, an expression cassette is cloned containing the therapeuticgene and its regulatory elements (Fig. 1). Packaging is achieved bytransfecting, using calcium phosphate co-precipitation, HEK 293cells with one plasmid containing the AAV vector and another plas-mid containing the AAV rep and cap genes without the ITRs. To stim-ulate the induction of cell permissivity to productive AAV replication,the cells are also infected with adenovirus or, more conveniently,treated with a third plasmid bearing the adenovirus helper genesE2A, E4 and VA-I RNA; the E1A and E1B genes are already expressedin the HEK 293 cells. Several laboratories now exploit a single helperplasmid, containing both the AAV2 rep and cap genes and the adeno-viral helper genes; in this case, the production of vectors involves celltransfection with only two plasmids [75,76]. Viral preparations arethen purified and concentrated from the cell lysates using cesiumchloride or iodixanol gradient centrifugation, or by chromatography[76]. These preparations are sufficiently pure to be used in both ex-perimental animals and in the clinics and have titers that can reachor surpass 1×1014 viral particles/ml; the concentration of viral parti-cles is thus several orders of magnitude higher than both VSV-G-pseudotyped retroviral vectors and adenoviral vectors.

The molecular determinants governing cell permissivity to effec-tive AAV transduction are still largely unclear. As a matter of fact,most AAV serotypes transduce post-mitotic cells in vivo, includingcardiomyocytes, skeletal myofibers, neurons, various cells in the

retina (ganglionar cells, pigment epithelium and photoreceptors) atvery high efficiency and, to a lesser extent, hepatocytes and cellsfrom the endocrine and exocrine pancreas. The reasons AAV is partic-ularly efficient in these cell types are still largely unknown; howeverthey clearly involve molecular events following vector internalizationinside the cells. A most likely scenario is that, in replicating cells thatare not permissive to AAV transduction, proteins of the cellular DNADamage Response (DDR) machinery (including members of theMre11–Rad50–Nbs1, MRN, complex) bind the AAV genomes andblock its conversion from single-stranded to double-stranded DNA[77–79]. The exquisite sensitivity of post-mitotic cells to AAV trans-duction would instead be explained by the downregulation of theseproteins upon terminal cell differentiation [80–82].

AAV vectors carrying a gene cassette cloned in the form of twocomplementary copies, positioned in tandem one after the other, nat-urally fold back into double-stranded, transcriptionally active DNA(self-complementary AAV vectors, scAAVs) and are thus significantlymore effective than linear AAV vectors [83,84]. Improvement is, how-ever, still marginal since regions of single-stranded DNA and thestructured sequences at the AAV hairpins are still substrates forDDR recognition, which in any case limit AAV transduction of repli-cating cells using these vectors (unpublished observations).

3. Lessons on gene therapy vectors learned from preclinicalexperimentation and clinical trials

The gene therapy clinical trial database maintained atWiley reportsthat, up to June 2011, over 1700 clinical trials have been conducted inover 30 countries worldwide (http://www.wiley.com/legacy/wileychi/genmed/clinical/). After an initial period of enthusiasm in the mid1990s, when the numbers of trials rose very rapidly to reach an averageof over 100 trials per year, the death of a patient enrolled in an experi-mentation for the deficit of ornithine-transcarbamylase using ad adeno-viral vector in 1999 [85], the development of leukemia in two patientswith SCID-X1 treated with a gammaretroviral vector (in 2002; [86])and, more in general, the growing perception of the general inefficacyof the protocols so far developed, determined a decline in the numberof experimentations in subsequent years. More recently, however, theenthusiasm for gene therapy has grown again, thanks to the success oftrials conducted by bone marrow gene transfer for inherited immuno-deficiency [87] and, most notably, an increasing number of applicationsexploiting the properties of AAV vectors for gene therapy of retinal [88]and neurodegenerative disorders [87]. Since the second half of the2000s, over 100 clinical trials were again registered in the regulatorydatabases worldwide, however, with a spectrum of applications signif-icantly different from those of the early days, which now include,among others, Parkinson's and Alzheimer's disease, retinal degenera-tions and heart failure. Less than 10% of the trials have addressed hered-itary monogenic diseases, conditions that initially inspired thedevelopment of gene therapy itself (reviewed in ref.: [2]); Fig. 2A.

Fig. 2. Human gene therapy clinical trials. The total number of gene therapy clinical tri-als since 1989 is shown according to therapeutic indication (A) and type of vector (B).Information is from the Wiley Gene Therapy Clinical Trials Worldwide.


Noticeably, more than two thirds of clinical studies so far con-ducted are based on the use of viral vectors for gene administration(Fig. 2B). Gammaretroviral vectors, which were the delivery systemof choice in the first half of the 1990s and overall constitute about20% of the trials conducted to date, have been much less consideredin recent years. This is due, in particular, to two major problems, thefirst related to the inability of these vectors to transduce non-replicating cells, and the second to their potential for insertional mu-tagenesis. The pre-integration complex of these viruses, which in-cludes the viral cDNA and a series of proteins of cellular and viralorigin, among which the integrase enzyme, remains in the cytosoland does not have access to the nucleus except during mitosis,when the nuclear membrane breaks down [89]. Since most of thecells in our body, including neurons, skeletal muscle cells, cardiomyo-cytes, endothelial cells and the vast majority of peripheral blood lym-phocytes, rarely divide or do not divide at all, the use ofgammaretroviral vectors is essentially restricted to ex vivo applica-tions on cells actively maintained in the cell cycle.

Regarding insertional mutagenesis due to gammaretroviral inser-tion into the human genome, a large series of studies has indeednow indicated that these viruses preferentially target, for their inser-tion, the promoter region of actively transcribed genes [90,91]. Theproblem of insertional mutagenesis was brought dramatically to theattention of the scientific and medical community in 2002 [92,93],when two children with X1-SCID (an inherited immunodeficiencydisease caused by the absence of the common gamma chain, a proteinencoded by the X chromosome that takes part in the formation of sev-eral interleukin receptors), treated in Paris by gammaretroviral genetherapy of hematopoietic stem cells, developed an acute lymphoblas-tic leukemia (T-ALL), which was later shown to be due to the inser-tion of the retroviral vector inside the LMO2 (LIM domain only 2)proto-oncogene [94,95]. Subsequently, another two children fromthe Paris cohort and one out of another ten treated in London also de-veloped T-ALL [96]. In all these patients, the LMO2 protein resultedoverexpressed due to retroviral insertional mutagenesis. Since thistime, insertional mutagenesis followed by preneoplastic or truly neo-plastic cell expansion has been observed in various other instances,including gene therapy of X-linked chronic granulomatous disease[97] and Wiskott–Aldrich Syndrome (WAS) [98], as well as a conse-quence of hematopoietic gene transfer in various animal models[99–101]. Of note, however, one of the most successful applicationsof gene therapy of hematopoietic stem cells so far, that foradenosine-deaminase deficiency [87], is also based on the use of gam-maretroviral vectors conceptually and structurally similar to those ofthe above mentioned trials, yet no evidence for clonal expansion ofthe transduced cells has ever been obtained [102]. This discrepancyis probably related to the role of the transgene itself in cooperatingwith cellular factors in driving selection and thus abnormal expansionof the transduced cells.

Since 2003, lentiviral vectors have entered the gene therapyarena, and have gained progressive popularity, especially due tothe property to transduce non-replicating cells, both in vivo and exvivo. The first clinical trial was approved for gene therapy of HIV-1infection [103]; since then, almost 40 other trials are ongoing orawaiting approval, including studies for different monogenic disor-ders (such as mucopolysaccharidosis, β-thalassemia, sickle cell ane-mia, X linked cerebral adrenoleukodystrophy, Fanconi anemia andX-SCID1), as well as those for various cancers (such as metastaticmelanoma, non-Hodgkin lymphoma and leukemia) and, more re-cently, for Parkinson's disease. While safety concerns raised by firstand second generation lentiviral vectors have been successfullyaddressed by the development of third generation vectors, in whichmost HIV-1 accessory proteins are not required for production,whetherintegration of these vectors into the host cell genomemight lead to theinappropriate activation of cellular genes through insertional mutagen-esis, similar to gammaretroviruses is still debated. Ex vivo cell

transduction indicates that these vectors, similar to wild type HIV-1,also integrate in correspondence with cellular transcribed genes [104].However, the region where integration occurs corresponds to thewhole gene transcription unit, in contrast to gammaretroviruses,which preferentially integrate in correspondencewith the transcriptionstart site, including the gene promoter and first intron [105]. A recentclinical trial for adrenoleukodystrophy (ALD) showed the efficacy andsafety of lentiviral gene transfer in hematopoietic stem progenitorcells [106]. However, several common insertion sites (CIS) were foundin the patients' cells, suggesting that lentiviral integrations conferred aselective advantage. However, high-throughput lentiviral vector inte-gration site analysis on human hematopoietic stem progenitor cellsengrafted in immunodeficient mice revealed the same CISs reportedin patients with ALD, mainly clustered in megabase-wide chromosomalregions. Overall, these findings might imply that lentiviral CISs are pro-duced by a benign integration bias toward specific genomic regionsrather than by oncogenic selection, as it occurs with gammaretro-viruses [107]. A most recent trial involving lentiviral β-globin genetransfer to an adult patient with severe β-thalassaemia showed suc-cessful correction of the disease at 21 months after gene therapy, withcomplete independence from transfusion [108]. Of note, most of thetherapeutic benefit resulted from a dominant, myeloid-biased cellclone, in which the integrated vector caused overexpression ofHMGA2, a protein that interacts with transcription factors to regulategene expression [108]. Whether the clonal dominance that accom-panies therapeutic efficacy was coincidental and stochastic or resultedfrom a hitherto benign cell expansion caused by deregulation of theHMGA2 gene in stem/progenitor cells, still remains an open question.

First and second generation adenoviral vectors, which have beenused in about 25% of clinical trials, particularly in the second half ofthe 1990s, now raise important safety concerns because of their pro-pensity to elicit important inflammatory and immune responses. Im-mediately after inoculation of these vectors in vivo, expression of aseries of inflammatory cytokines is activated, determining recruit-ment, to the sites of inoculation, of macrophages, neutrophils andNK cells [109]. For example, in the liver, 80–90% of vector is rapidlyeliminated by this inflammatory response within the first 24 h afterinoculation [110]. This response is triggered by the adenoviral particleitself, and does not require viral gene expression. The powerful induc-tion of an inflammatory and immune response was the cause of deathin an 18-year old patient enrolled in a gene therapy clinical trial forthe hereditary deficit of ornithine transcarbamylase (OTC), an enzymeof the urea cycle, at the University of Pennsylvania, Philadelphia, PA in1999 [111,112]. Subsequently, starting from 4 to 7 days after injection,the antibody and cellular immune response become activated. Theinoculation site is infiltrated by cytotoxic T lymphocytes, which recog-nize and eliminate the transduced cells. Furthermore, the immune sys-tem mounts a very vigorous antibody response, which, thanks to theproduction of neutralizing antibodies, prevents any possibility of re-injecting the same vector or vectors based on the same serotype. Inthis context, it is also important to observe that 30–40% of individualsliving in western countries and 80–90% of those living in sub-SaharanAfrica naturally possess anti-Adenovirus serotype 5 antibodies, whichcompletely prevent utilization of this serotype for gene therapy or vac-cination [113].

In light of the above observations, the utilization of first and sec-ond generation adenoviral vectors should now be limited to applica-tions in which prolonged transgene expression is not desirable orrequired, and in which immune stimulation is instead a requisite. Inpractical terms, this is the case in two very important applications:gene therapy of cancer and genetic vaccination.

In the case of gutless vectors, the systemic administration of theseviruses continues to stimulate the immune response, similar to firstand second generation vectors, since this depends on the viral capsidproteins [114]. The same proteins also trigger the production of neu-tralizing antibodies, which prevent re-administration of vectors of the


same serotype. After the initial inflammation, however, the gutlessvectors do not express any viral genes, and the transduced cells aretherefore not recognized and eliminated by the immune system, un-less the transgene protein itself is immunogenic. Despite the great po-tential of gutless vectors, however, their clinical application is stilllimited by two major technical issues; contamination with a still un-acceptable proportion of helper virus and the difficulty to obtain thelarge batches of vectors that are needed for clinical use. From a clini-cal perspective, it is worth mentioning the possibility to express verylong genes, which would not fit the cloning capacity of most othervector types. For instance, one patient suffering from severe hemo-philia A has been treated with a gutless adenoviral vector expressingfull-length Factor VIII, which resulted in 1% of normal Factor VIIIlevels for several months. However, a transient inflammatory re-sponse with hematologic and liver abnormalities was observed andno additional patients were recruited in the study [115].

While the strong immunological and inflammatory response eli-cited by adenoviral vectors currently limits their clinical utilization,over the last 10 years there has been a growing interest in the useof adenovirus mutants designed to replicate exclusively in tumorcells (conditionally replicative adenovirus, CRAd) for lytic virotherapyof cancer. A first oncolytic adenovirus, named ONYX-015, was devel-oped by deleting the gene coding for E1B-55K from the wild type viralgenome, based on the observation that the E1B-deleted viruses werereplicating well in cells lacking the p53 oncosuppressor (as it occursin ~50% of human cancers), but not in untransformed cells, havingwild type p53 [116]. This E1B-mutated virus was originally used inPhase I and II clinical trials based on the intratumoral injection ofthe virus in patients with recurrent head and neck cancer [117]. Fol-lowing these trials, a number of additional experimentations have fol-lowed in the subsequent years [118]. The available results haveshown that the beneficial effects of the ONYX-015 virus were best ap-preciable in conjunction with conventional chemotherapy [119]. Fur-ther studies then indicated that the tumor selectivity of this virus wasprobably independent of p53 function, and is currently mainly attrib-uted to late mRNA export (reviewed in ref.: [120])

Other clinical trials are currently ongoing using a new generation ofoncolytic adenoviruses, in which E1B is deleted and E1A is under thetranscriptional control of a tissue-specific promoter (in particular, thepromoter of the prostate antigen PSA), in order to achieve selectiveviral replication in specific cell types [121–123], or in which E1A carriesmutations improving viral replication in cancer cells [124,125]. In addi-tion, a third generation of conditionally replicative adenoviruses areunder development, also delivering therapeutic genes (the so called“armed” oncolytic adenoviruses), including cytokines, shRNAs, or fac-tors that increase virus spread (reviewed in refs.: [126,127]).

AAV vectors represent an outstanding tool for in vivo gene trans-fer for a series of reasons, which include their genetic simplicity (noviral protein is expressed in the target cells; therefore, these vectorsare not immunogenic and do not cause inflammation), the lack of in-tegration into the host cell genome, while they persist in an episomalform, probably as head-to-tail or head-to-head extrachromosomalconcatamers, in non-replicating cells and, in the long term, persis-tence in the transduced animals, virtually coinciding with the wholelife of the animal, at least in short-lived rodents [128]. Due to thesevery favorable characteristics, the clinical utilization of AAV vectorshas risen substantially in the last few years, with over 80 trials con-ducted. These are Phase I/II trials for various hereditary (in particular,hemophilia B, deficit of α1-antitripsin, cystic fibrosis, muscular dys-trophies, retinal degeneration) and acquired (rheumatoid arthritis,Parkinson's disease, Alzheimer's disease, heart failure) disorders (ex-tensively discussed in ref: [2]). Outstanding clinical success has beenso far achieved in a few of these trials, most notably those for aninherited form of blindness due to a defect of the visual cycle proteinRPE-65 [129,130], while promising results are currently obtained byothers, most notably in one trial for heart failure [131].

A few of the clinical trials have addressed therapy of hemophilia B,an inherited disorder with a prevalence of 1:25,000 males, due to thedeficiency of coagulation Factor IX. In both knock-out mice and thehemophilic dog, AAV has proven very effective in correcting the de-fect and has shown permanent restoration of normal coagulation ac-tivity [132]. Based on these promising findings, a first phase I/IIclinical trial was started in 2000 as an open label, dose-escalationstudy, entailing the intramuscular injections of an AAV2 vector ex-pressing human Factor IX [133,134]. A few years later, the sameAAV2 vector expressing human Factor IX was infused into the liverthrough the hepatic artery in three dose cohorts of subjects with se-vere hemophilia B [135]. At the highest dose, one of the patientsshowed levels of circulating Factor IX higher than 10% of normal,peaking at 2 weeks after injection and persisting for at least 4 weeks[135]. However, in contrast to that observed in the animal models,the production of the factor progressively decreased until it becameundetectable 14 weeks after treatment. Apparently, this unexpectedoccurrence was not due to the presence of anti-Factor IX antibodies,but rather to the development of an immune response against theAAV vector capsid proteins, by which the transduced hepatocyteswere eliminated by the patient's CD8+ lymphocytes [135]. Such anoccurrence, however, has not been observed in animal models and,in addition, in this trial occurred relatively late after vector inocula-tion. One explanation brought forward for these findings is thatAAV2 is a common infectious agent for humans but not for other an-imal species, and thus transduction might have reactivated to preex-isting immune recognition of the surface protein of the virus, whilethe delayed kinetics with which the immune response reacted toviral inoculation might be related to the prolonged persistence ofthe AAV capsid proteins in the transduced cells [136].

Whether these explanations might satisfactorily explain the re-sults, and to what extent previous immune activation against AAVmight hamper the successful use of these vectors in the clinics, how-ever, will await the execution of further clinical experimentations. Inthe meantime, other trials exploiting AAV vectors in patients considerthe inclusion of a transient period of immunosuppression immediate-ly after AAV vector inoculation, in order to avoid immune recognitionof the viral capsid proteins [137].

4. Conclusions

Considering all the above issues, it can safely be concluded thatgene therapy continues to be conceptually very exciting and medical-ly appealing, since it has the potential to treat or even cure severaldiseases that are beyond the reach of chemical drugs or conventionaltherapeutic modalities. Nevertheless it is equally evident that movinggene therapy towards its current state has been remarkably challeng-ing, and progress has been slow to come. A large part of the interven-ing difficulties have been in the field of gene delivery itself, whereovercoming the barrier imposed by the chemical nature of nucleicacids on one hand and the cell membrane on the other still representsa difficult problem to solve. It is in this perspective that viral deliverymethods currently represent by far the most appealing option for ef-ficient gene delivery both ex vivo and in vivo. Should we envisage afuture in which we still rely on modified viruses to obtain nucleicacid internalization into the target cells? Probably not, and this willbe most welcome in light of the intrinsic problems related to the safe-ty, complexity and undesired effect that viruses elicit. Nevertheless,we still havemuch to learn from viruses since they are optimized in vir-tually all steps necessary for efficient gene delivery, including nucleicacid protection and transport in the extracellular environment, target-ing of specific receptors, internalization and routing of nucleic acids tonucleus and, in some instances, permanent modification of the hostcell genome. Imitating some of these features, albeit not necessarilyusing intact virions, will contribute to progress in the field towardsbroader and more successful application.


Acknowledgments

The authors are grateful to Suzanne Kerbavcic for excellent edito-rial assistance.

This work was supported by grant GGP11068 from the TelethonFoundation, Italy, by the Advanced Grant 20090506 from the EuropeanResearch Council (ERC) and by Project CTC from the FondazioneCRTrieste, Trieste, Italy.

References

[1] J. Couzin-Frankel, Genetics. The promise of a cure: 20 years and counting, Sci-ence 324 (2009) 1504–1507.

[2] M. Giacca, Gene Therapy, Springer978-88-470-1642-2, 2010 pp. 1–309.[3] A.D. Miller, Retroviral vectors, Curr. Top. Microbiol. Immunol. 158 (1992) 1–24.[4] R.L. Friedman, Expression of human adenosine deaminase using a transmissable

murine retrovirus vector system, Proc. Natl. Acad. Sci. U. S. A. 82 (1985) 703–707.[5] A.D. Miller, Retrovirus packaging cells, Hum. Gene Ther. 1 (1990) 5–14.[6] C. Aiken, Pseudotyping human immunodeficiency virus type 1 (HIV-1) by the

glycoprotein of vesicular stomatitis virus targets HIV-1 entry to an endocyticpathway and suppresses both the requirement for Nef and the sensitivity to cy-closporin A, J. Virol. 71 (1997) 5871–5877.

[7] J.K. Yee, T. Friedmann, J.C. Burns, Generation of high-titer pseudotyped retroviralvectors with very broad host range, Methods Cell Biol. 43 (1994) 99–112.

[8] T. Kimura, R.C. Koya, L. Anselmi, C. Sternini, H.J. Wang, B. Comin-Anduix, R.M.Prins, E. Faure-Kumar, N. Rozengurt, Y. Cui, N. Kasahara, R. Stripecke, Lentiviralvectors with CMV or MHCII promoters administered in vivo: immune reactivityversus persistence of expression, Mol. Ther. 15 (2007) 1390–1399.

[9] D.C. Carpentier, K. Vevis, A. Trabalza, C. Georgiadis, S.M. Ellison, R.I. Asfahani, N.D.Mazarakis, Enhanced pseudotyping efficiency of HIV-1 lentiviral vectors by a rabies/vesicular stomatitis virus chimeric envelope glycoprotein, Gene Ther. (In press).

[10] S. Kato, M. Kuramochi, K. Takasumi, K. Kobayashi, K.I. Inoue, D. Takahara, S.Hitoshi, K. Ikenaka, T. Shimada, M. Takada, Neuron-specific gene transferthrough retrograde transport of lentiviral vector pseudotyped with a noveltype of fusion envelope glycoprotein, Hum. Gene Ther. 22 (2011) 1511–1523.

[11] A. Muik, I. Kneiske, M. Werbizki, D. Wilflingseder, T. Giroglou, O. Ebert, A. Kraft,U. Dietrich, G. Zimmer, S. Momma, D. von Laer, Pseudotyping vesicular stomati-tis virus with lymphocytic choriomeningitis virus glycoproteins enhances infec-tivity for glioma cells and minimizes neurotropism, J. Virol. 85 (2011)5679–5684.

[12] H.G. Yang, B.L. Hu, L. Xiao, P. Wang, Dendritic cell-directed lentivector vaccineinduces antigen-specific immune responses against murine melanoma, CancerGene Ther. 18 (2011) 370–380.

[13] S. Froelich, A. Tai, K. Kennedy, A. Zubair, P. Wang, Pseudotyping lentiviral vectorswith aura virus envelope glycoproteins for DC-SIGN-mediated transduction ofdendritic cells, Hum. Gene Ther. 22 (2011) 1281–1291.

[14] Y. Suzuki, R. Craigie, The road to chromatin — nuclear entry of retroviruses, Nat.Rev. Microbiol. 5 (2007) 187–196.

[15] A.H. Chang, M. Sadelain, The genetic engineering of hematopoietic stem cells:the rise of lentiviral vectors, the conundrum of the ltr, and the promise oflineage-restricted vectors, Mol. Ther. 15 (2007) 445–456.

[16] A. Schambach, C. Baum, Clinical application of lentiviral vectors — concepts andpractice, Curr. Gene Ther. 8 (2008) 474–482.

[17] L. Naldini, U. Blomer, P. Gallay, D. Ory, R. Mulligan, F.H. Gage, I.M. Verma, D.Trono, In vivo gene delivery and stable transduction of nondividing cells by alentiviral vector, Science 272 (1996) 263–267.

[18] P.L. Sinn, S.L. Sauter, P.B. McCray Jr., Gene therapy progress and prospects: de-velopment of improved lentiviral and retroviral vectors — design, biosafety,and production, Gene Ther. 12 (2005) 1089–1098.

[19] P.Manilla, T. Rebello, C. Afable, X. Lu, V. Slepushkin, L.M.Humeau, K. Schonely, Y. Ni,G.K. Binder, B.L. Levine, R.R. MacGregor, C.H. June, B. Dropulic, Regulatory con-siderations for novel gene therapy products: a review of the process leading tothe first clinical lentiviral vector, Hum. Gene Ther. 16 (2005) 17–25.

[20] R. Zufferey, T. Dull, R.J. Mandel, A. Bukovsky, D. Quiroz, L. Naldini, D. Trono, Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery, J. Virol.72 (1998) 9873–9880.

[21] M. Manganini, M. Serafini, F. Bambacioni, C. Casati, E. Erba, A. Follenzi, L. Naldini,S. Bernasconi, G. Gaipa, A. Rambaldi, A. Biondi, J. Golay, M. Introna, A human im-munodeficiency virus type 1 pol gene-derived sequence (cPPT/CTS) increasesthe efficiency of transduction of human nondividing monocytes and T lympho-cytes by lentiviral vectors, Hum. Gene Ther. 13 (2002) 1793–1807.

[22] T. VandenDriessche, L. Thorrez, L. Naldini, A. Follenzi, L. Moons, Z. Berneman,D. Collen, M.K. Chuah, Lentiviral vectors containing the human immunodefi-ciency virus type-1 central polypurine tract can efficiently transduce nondi-viding hepatocytes and antigen-presenting cells in vivo, Blood 100 (2002)813–822.

[23] L.K. Law, B.L. Davidson, What does it take to bind CAR? Mol. Ther. 12 (2005)599–609.

[24] X. Danthinne, M.J. Imperiale, Production of first generation adenovirus vectors: areview, Gene Ther. 7 (2000) 1707–1714.

[25] W.C. Russell, Adenoviruses: update on structure and function, J. Gen. Virol. 90(2009) 1–20.

[26] N. Bessis, F.J. GarciaCozar, M.C. Boissier, Immune responses to gene therapy vec-tors: influence on vector function and effector mechanisms, Gene Ther. 11(Suppl. 1) (2004) S10–S17.

[27] N. Chirmule, K. Propert, S. Magosin, Y. Qian, R. Qian, J. Wilson, Immune re-sponses to adenovirus and adeno-associated virus in humans, Gene Ther. 6(1999) 1574–1583.

[28] Q. Liu, D.A. Muruve, Molecular basis of the inflammatory response to adenovirusvectors, Gene Ther. 10 (2003) 935–940.

[29] Q. Wang, M.H. Finer, Second-generation adenovirus vectors, Nat. Med. 2 (1996)714–716.

[30] R. Alba, A. Bosch, M. Chillon, Gutless adenovirus: last-generation adenovirus forgene therapy, Gene Ther. 12 (Suppl. 1) (2005) S18–S27.

[31] N. Brunetti-Pierri, P. Ng, Progress and prospects: gene therapy for genetic diseaseswith helper-dependent adenoviral vectors, Gene Ther. 15 (2008) 553–560.

[32] R.J. Parks, L. Chen, M. Anton, U. Sankar, M.A. Rudnicki, F.L. Graham, A helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal, Proc. Natl. Acad. Sci. U. S. A. 93(1996) 13565–13570.

[33] R. Argnani, M. Lufino, M. Manservigi, R. Manservigi, Replication-competent her-pes simplex vectors: design and applications, Gene Ther. 12 (Suppl. 1) (2005)S170–S177.

[34] A.R. Frampton Jr., W.F. Goins, K. Nakano, E.A. Burton, J.C. Glorioso, HSV traffick-ing and development of gene therapy vectors with applications in the nervoussystem, Gene Ther. 12 (2005) 891–901.

[35] T.C. Liu, D. Kirn, Gene therapy progress and prospects cancer: oncolytic viruses,Gene Ther. 15 (2008) 877–884.

[36] R. Kanai, H. Wakimoto, T. Cheema, S.D. Rabkin, Oncolytic herpes simplex virusvectors and chemotherapy: are combinatorial strategies more effective for can-cer? Future Oncol. 6 (2010) 619–634.

[37] A.L. Epstein, P. Marconi, R. Argnani, R. Manservigi, HSV-1-derived recombinantand amplicon vectors for gene transfer and gene therapy, Curr. Gene Ther. 5(2005) 445–458.

[38] H.L. Kaufman, S.D. Bines, OPTIM trial: a Phase III trial of an oncolytic herpes virusencoding GM-CSF for unresectable stage III or IV melanoma, Future Oncol. 6(2010) 941–949.

[39] S.K. Geevarghese, D.A. Geller, H.A. de Haan, M. Horer, A.E. Knoll, A. Mescheder, J.Nemunaitis, T.R. Reid, D.Y. Sze, K.K. Tanabe, H. Tawfik, Phase I/II study of oncolyticherpes simplex virus NV1020 in patients with extensively pretreated refractorycolorectal cancer metastatic to the liver, Hum. Gene Ther. 21 (2010) 1119–1128.

[40] K.J. Harrington, M. Hingorani, M.A. Tanay, J. Hickey, S.A. Bhide, P.M. Clarke, L.C.Renouf, K. Thway, A. Sibtain, I.A. McNeish, K.L. Newbold, H. Goldsweig, R.Coffin, C.M. Nutting, Phase I/II study of oncolytic HSV GM-CSF in combinationwith radiotherapy and cisplatin in untreated stage III/IV squamous cell cancerof the head and neck, Clin. Cancer Res. 16 (2010) 4005–4015.

[41] J.C. Glorioso, D.J. Fink, Herpes vector-mediated gene transfer in treatment of dis-eases of the nervous system, Annu. Rev. Microbiol. 58 (2004) 253–271.

[42] E.A. Burton, D.J. Fink, J.C. Glorioso, Replication-defective genomic HSV gene ther-apy vectors: design, production and CNS applications, Curr. Opin. Mol. Ther. 7(2005) 326–336.

[43] A.L. Epstein, Progress and prospects: biological properties and technological ad-vances of herpes simplex virus type 1-based amplicon vectors, Gene Ther. 16(2009) 709–715.

[44] P. Marconi, R. Manservigi, A.L. Epstein, HSV-1-derived helper-independent de-fective vectors, replicating vectors and amplicon vectors, for the treatment ofbrain diseases, Curr. Opin. Drug Discov. Devel. 13 (2010) 169–183.

[45] D.S. Latchman, Herpes simplex virus vectors for gene delivery to a variety of dif-ferent cell types, Curr. Gene Ther. 2 (2002) 415–426.

[46] R. Ferrera, D. Cuchet, C. Zaupa, V. Revol-Guyot, M. Ovize, A.L. Epstein, Efficientand non-toxic gene transfer to cardiomyocytes using novel generation ampliconvectors derived from HSV-1, J. Mol. Cell. Cardiol. 38 (2005) 219–223.

[47] B. Cao, J. Huard, Gene transfer to skeletal muscle using herpes simplex virus-based vectors, Methods Mol. Biol. 246 (2004) 301–308.

[48] P. Marconi, R. Argnani, A.L. Epstein, R. Manservigi, HSV as a vector in vaccine de-velopment and gene therapy, Adv. Exp. Med. Biol. 655 (2009) 118–144.

[49] K.I. Berns, R.M. Linden, The cryptic life style of adeno-associated virus, Bioessays17 (1995) 237–245.

[50] K. Erles, P. Sebokova, J.R. Schlehofer, Update on the prevalence of serum antibodies(IgG and IgM) to adeno-associated virus (AAV), J. Med. Virol. 59 (1999) 406–411.

[51] K.I. Berns, Parvovirus replication, Microbiol. Rev. 54 (1990) 316–329.[52] Z. Wu, A. Asokan, R.J. Samulski, Adeno-associated virus serotypes: vector toolkit

for human gene therapy, Mol. Ther. 14 (2006) 316–327.[53] L. Zentilin, M. Giacca, Adeno-associated virus vectors: versatile tools for in vivo

gene transfer, Contrib. Nephrol. 159 (2008) 63–77.[54] C. Summerford, J.S. Bartlett, R.J. Samulski, AlphaVbeta5 integrin: a co-receptor

for adeno-associated virus type 2 infection, Nat. Med. 5 (1999) 78–82.[55] K. Qing, C. Mah, J. Hansen, S. Zhou, V. Dwarki, A. Srivastava, Human fibroblast

growth factor receptor 1 is a co-receptor for infection by adeno-associatedvirus 2, Nat. Med. 5 (1999) 71–77.

[56] J.E. Rabinowitz, D.E. Bowles, S.M. Faust, J.G. Ledford, S.E. Cunningham, R.J.Samulski, Cross-dressing the virion: the transcapsidation of adeno-associated virus serotypes functionally defines subgroups, J. Virol. 78 (2004)4421–4432.

[57] A.J. Lotery, G.S. Yang, R.F. Mullins, S.R. Russell, M. Schmidt, E.M. Stone, J.D.Lindbloom, J.A. Chiorini, R.M. Kotin, B.L. Davidson, Adeno-associated virus type5: transduction efficiency and cell-type specificity in the primate retina, Hum.Gene Ther. 14 (2003) 1663–1671.


[58] Z. Wu, E. Miller, M. Agbandje-McKenna, R.J. Samulski, Alpha2,3 and alpha2,6N-linked sialic acids facilitate efficient binding and transduction by adeno-associated virus types 1 and 6, J. Virol. 80 (2006) 9093–9103.

[59] J.A. Chiorini, L. Yang, Y. Liu, B. Safer, R.M. Kotin, Cloning of adeno-associatedvirus type 4 (AAV4) and generation of recombinant AAV4 particles, J. Virol. 71(1997) 6823–6833.

[60] B. Akache, D. Grimm, K. Pandey, S.R. Yant, H. Xu, M.A. Kay, The 37/67-kilodaltonlaminin receptor is a receptor for adeno-associated virus serotypes 8, 2, 3, and 9,J. Virol. 80 (2006) 9831–9836.

[61] D. Duan, Q. Li, A.W. Kao, Y. Yue, J.E. Pessin, J.F. Engelhardt, Dynamin is requiredfor recombinant adeno-associated virus type 2 infection, J. Virol. 73 (1999)10371–10376.

[62] U. Bantel-Schaal, B. Hub, J. Kartenbeck, Endocytosis of adeno-associated virustype 5 leads to accumulation of virus particles in the Golgi compartment, J. Virol.76 (2002) 2340–2349.

[63] J.S. Bartlett, R.Wilcher, R.J. Samulski, Infectious entry pathway of adeno-associatedvirus and adeno-associated virus vectors, J. Virol. 74 (2000) 2777–2785.

[64] B. Hauck, W. Xiao, Characterization of tissue tropism determinants of adeno-associated virus type 1, J. Virol. 77 (2003) 2768–2774.

[65] M. Weber, J. Rabinowitz, N. Provost, H. Conrath, S. Folliot, D. Briot, Y. Cherel, P.Chenuaud, J. Samulski, P. Moullier, F. Rolling, Recombinant adeno-associatedvirus serotype 4 mediates unique and exclusive long-term transduction of reti-nal pigmented epithelium in rat, dog, and nonhuman primate after subretinaldelivery, Mol. Ther. 7 (2003) 774–781.

[66] H. Cheng, S.H. Wolfe, V. Valencia, K. Qian, L. Shen, M.I. Phillips, L.J. Chang, Y.C.Zhang, Efficient and persistent transduction of exocrine and endocrine pancreasby adeno-associated virus type 8, J. Biomed. Sci. 14 (2007) 585–594.

[67] V. Jimenez, E. Ayuso, C. Mallol, J. Agudo, A. Casellas, M. Obach, S. Munoz, A.Salavert, F. Bosch, In vivo genetic engineering of murine pancreatic beta cellsmediated by single-stranded adeno-associated viral vectors of serotypes 6,8 and 9, Diabetologia 54 (2011) 1075–1086.

[68] K. Inagaki, S. Fuess, T.A. Storm, G.A. Gibson, C.F. McTiernan, M.A. Kay, H. Nakai,Robust systemic transduction with AAV9 vectors in mice: efficient global cardiacgene transfer superior to that of AAV8, Mol. Ther. 14 (2006) 45–53.

[69] J.N. Kornegay, J. Li, J.R. Bogan, D.J. Bogan, C. Chen, H. Zheng, B. Wang, C. Qiao, J.F.Howard Jr., X. Xiao, Widespread muscle expression of an AAV9 human mini-dystrophin vector after intravenous injection in neonatal dystrophin-deficientdogs, Mol. Ther. 18 (2010) 1501–1508.

[70] R.M. Linden, E. Winocour, K.I. Berns, The recombination signals for adeno-associated virus site-specific integration, Proc. Natl. Acad. Sci. U. S. A. 93(1996) 7966–7972.

[71] N. Dutheil, F. Shi, T. Dupressoir, R.M. Linden, Adeno-associated virus site-specifically integrates into a muscle-specific DNA region, Proc. Natl. Acad. Sci.U. S. A. 97 (2000) 4862–4866.

[72] R.M. Kotin, R.M. Linden, K.I. Berns, Characterization of a preferred site on humanchromosome 19q for integration of adeno-associated virus DNA by non-homologous recombination, EMBO J. 11 (1992) 5071–5078.

[73] R.H. Smith, Adeno-associated virus integration: virus versus vector, Gene Ther.15 (2008) 817–822.

[74] L. Tenenbaum, E. Lehtonen, P.E. Monahan, Evaluation of risks related to the useof adeno-associated virus-based vectors, Curr. Gene Ther. 3 (2003) 545–565.

[75] D. Grimm, A. Kern, K. Rittner, J.A. Kleinschmidt, Novel tools for production andpurification of recombinant adenoassociated virus vectors, Hum. Gene Ther. 9(1998) 2745–2760.

[76] M. Mezzina, O.W. Merten, Adeno-associated viruses, Methods Mol. Biol. 737(2011) 211–234.

[77] F.K. Ferrari, T. Samulski, T. Shenk, R.J. Samulski, Second-strand synthesis is arate-limiting step for efficient transduction by recombinant adeno-associatedvirus vectors, J. Virol. 70 (1996) 3227–3234.

[78] K.J. Fisher, G.P. Gao, M.D. Weitzman, R. DeMatteo, J.F. Burda, J.M. Wilson, Trans-duction with recombinant adeno-associated virus for gene therapy is limited byleading-strand synthesis, J. Virol. 70 (1996) 520–532.

[79] L. Zentilin, A. Marcello, M. Giacca, Involvement of cellular double-strand DNAbreak-binding proteins in processing of recombinant adeno-associated virus(AAV) genome, J. Virol. 75 (2001) 12279–12287.

[80] T. Cervelli, J.A. Palacios, L. Zentilin, M. Mano, R.A. Schwartz, M.D. Weitzman, M.Giacca, Processing of recombinant AAV genomes occurs in specific nuclear struc-tures that overlap with foci of DNA-damage-response proteins, J. Cell Sci. 121(2008) 349–357.

[81] C.A. Adelman, S. De, J.H. Petrini, Rad50 is dispensable for the maintenance andviability of postmitotic tissues, Mol. Cell. Biol. 29 (2009) 483–492.

[82] M. Simonatto, L. Latella, P.L. Puri, DNA damage and cellular differentiation: morequestions than responses, J. Cell. Physiol. 213 (2007) 642–648.

[83] M.L. Hirsch, L. Green, M.H. Porteus, R.J. Samulski, Self-complementary AAV me-diates gene targeting and enhances endonuclease delivery for double-strandbreak repair, Gene Ther. 17 (2010) 1175–1180.

[84] D.M. McCarty, Self-complementary AAV vectors; advances and applications,Mol. Ther. 16 (2008) 1648–1656.

[85] E. Marshall, Gene therapy death prompts review of adenovirus vector, Science286 (1999) 2244–2245.

[86] R.H. Buckley, Gene therapy for SCID — a complication after remarkable progress,Lancet 360 (2002) 1185–1186.

[87] A. Aiuti, F. Cattaneo, S. Galimberti, U. Benninghoff, B. Cassani, L. Callegaro,S. Scaramuzza, G. Andolfi, M. Mirolo, I. Brigida, A. Tabucchi, F. Carlucci, M. Eibl,M. Aker, S. Slavin, H. Al-Mousa, A. Al Ghonaium, A. Ferster, A. Duppenthaler,L. Notarangelo, U.Wintergerst, R.H. Buckley,M. Bregni, S. Marktel, M.G. Valsecchi,

P. Rossi, F. Ciceri, R.Miniero, C. Bordignon,M.G. Roncarolo, Gene therapy for immu-nodeficiency due to adenosine deaminase deficiency, N. Engl. J. Med. 360 (2009)447–458.

[88] P.K. Buch, J.W. Bainbridge, R.R. Ali, AAV-mediated gene therapy for retinal disor-ders: from mouse to man, Gene Ther. 15 (2008) 849–857.

[89] D.G. Miller, M.A. Adam, A.D. Miller, Gene transfer by retrovirus vectors occursonly in cells that are actively replicating at the time of infection, Mol. Cell.Biol. 10 (1990) 4239–4242.

[90] C. Cattoglio, D. Pellin, E. Rizzi, G. Maruggi, G. Corti, F. Miselli, D. Sartori, A.Guffanti, C. Di Serio, A. Ambrosi, G. De Bellis, F. Mavilio, High-definition mappingof retroviral integration sites identifies active regulatory elements in humanmultipotent hematopoietic progenitors, Blood 116 (2010) 5507–5517.

[91] R. Daniel, J.A. Smith, Integration site selection by retroviral vectors: molecularmechanism and clinical consequences, Hum. Gene Ther. 19 (2008) 557–568.

[92] S. Hacein-Bey-Abina, C. von Kalle, M. Schmidt, F. Le Deist, N. Wulffraat, E.McIntyre, I. Radford, J.L. Villeval, C.C. Fraser, M. Cavazzana-Calvo, A. Fischer, Aserious adverse event after successful gene therapy for X-linked severe com-bined immunodeficiency, N. Engl. J. Med. 348 (2003) 255–256.

[93] S. Hacein-Bey-Abina, C. Von Kalle, M. Schmidt, M.P. McCormack, N. Wulffraat, P.Leboulch, A. Lim, C.S. Osborne, R. Pawliuk, E. Morillon, R. Sorensen, A. Forster, P.Fraser, J.I. Cohen, G. de Saint Basile, I. Alexander, U. Wintergerst, T. Frebourg, A.Aurias, D. Stoppa-Lyonnet, S. Romana, I. Radford-Weiss, F. Gross, F. Valensi, E.Delabesse, E. Macintyre, F. Sigaux, J. Soulier, L.E. Leiva, M. Wissler, C. Prinz, T.H.Rabbitts, F. Le Deist, A. Fischer, M. Cavazzana-Calvo, LMO2-associated clonal Tcell proliferation in two patients after gene therapy for SCID-X1, Science 302(2003) 415–419.

[94] D.B. Kohn, M. Sadelain, J.C. Glorioso, Occurrence of leukaemia following genetherapy of X-linked SCID, Nat. Rev. Cancer 3 (2003) 477–488.

[95] M.P. McCormack, T.H. Rabbitts, Activation of the T-cell oncogene LMO2 aftergene therapy for X-linked severe combined immunodeficiency, N. Engl. J. Med.350 (2004) 913–922.

[96] S. Hacein-Bey-Abina, A. Garrigue, G.P. Wang, J. Soulier, A. Lim, E. Morillon, E.Clappier, L. Caccavelli, E. Delabesse, K. Beldjord, V. Asnafi, E. MacIntyre, L. DalCortivo, I. Radford, N. Brousse, F. Sigaux, D. Moshous, J. Hauer, A. Borkhardt,B.H. Belohradsky, U. Wintergerst, M.C. Velez, L. Leiva, R. Sorensen, N.Wulffraat, S. Blanche, F.D. Bushman, A. Fischer, M. Cavazzana-Calvo, Insertionaloncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1, J.Clin. Invest. 118 (2008) 3132–3142.

[97] M.G. Ott, M. Schmidt, K. Schwarzwaelder, S. Stein, U. Siler, U. Koehl, H. Glimm, K.Kuhlcke, A. Schilz, H. Kunkel, S. Naundorf, A. Brinkmann, A. Deichmann, M.Fischer, C. Ball, I. Pilz, C. Dunbar, Y. Du, N.A. Jenkins, N.G. Copeland, U. Luthi,M. Hassan, A.J. Thrasher, D. Hoelzer, C. von Kalle, R. Seger, M. Grez, Correctionof X-linked chronic granulomatous disease by gene therapy, augmented by in-sertional activation of MDS1-EVI1, PRDM16 or SETBP1, Nat. Med. 12 (2006)401–409.

[98] K. Boztug, M. Schmidt, A. Schwarzer, P.P. Banerjee, I.A. Diez, R.A. Dewey, M.Bohm, A. Nowrouzi, C.R. Ball, H. Glimm, S. Naundorf, K. Kuhlcke, R. Blasczyk, I.Kondratenko, L. Marodi, J.S. Orange, C. von Kalle, C. Klein, Stem-cell gene thera-py for the Wiskott–Aldrich syndrome, N. Engl. J. Med. 363 (2010) 1918–1927.

[99] C. Cattoglio, G. Facchini, D. Sartori, A. Antonelli, A. Miccio, B. Cassani, M. Schmidt,C. von Kalle, S. Howe, A.J. Thrasher, A. Aiuti, G. Ferrari, A. Recchia, F. Mavilio, Hotspots of retroviral integration in human CD34+ hematopoietic cells, Blood 110(2007) 1770–1778.

[100] U.P. Dave, N.A. Jenkins, N.G. Copeland, Gene therapy insertional mutagenesis in-sights, Science 303 (2004) 333.

[101] G.D. Trobridge, Genotoxicity of retroviral hematopoietic stem cell gene therapy,Expert Opin. Biol. Ther. 11 (2011) 581–593.

[102] A. Aiuti, B. Cassani, G. Andolfi, M. Mirolo, L. Biasco, A. Recchia, F. Urbinati, C.Valacca, S. Scaramuzza, M. Aker, S. Slavin, M. Cazzola, D. Sartori, A. Ambrosi, C.Di Serio, M.G. Roncarolo, F. Mavilio, C. Bordignon, Multilineage hematopoieticreconstitution without clonal selection in ADA-SCID patients treated withstem cell gene therapy, J. Clin. Invest. 117 (2007) 2233–2240.

[103] R.R. MacGregor, Clinical protocol. A phase 1 open-label clinical trial of the safetyand tolerability of single escalating doses of autologous CD4 T cells transducedwith VRX496 in HIV-positive subjects, Hum. Gene Ther. 12 (2001) 2028–2029.

[104] A. Cereseto, M. Giacca, Integration site selection by retroviruses, AIDS Rev. 6(2004) 13–20.

[105] R. Daniel, J.A. Smith, Integration site selection by retroviral vectors: molecularmechanism and clinical consequences, Hum. Gene Ther. 19 (2008) 557–568.

[106] N. Cartier, S. Hacein-Bey-Abina, C.C. Bartholomae, G. Veres, M. Schmidt, I.Kutschera, M. Vidaud, U. Abel, L. Dal-Cortivo, L. Caccavelli, N. Mahlaoui, V.Kiermer, D. Mittelstaedt, C. Bellesme, N. Lahlou, F. Lefrere, S. Blanche, M. Audit,E. Payen, P. Leboulch, B. l'Homme, P. Bougneres, C. Von Kalle, A. Fischer, M.Cavazzana-Calvo, P. Aubourg, Hematopoietic stem cell gene therapy with a len-tiviral vector in X-linked adrenoleukodystrophy, Science 326 (2009) 818–823.

[107] A. Biffi, C.C. Bartolomae, D. Cesana, N. Cartier, P. Aubourg, M. Ranzani, M. Cesani,F. Benedicenti, T. Plati, E. Rubagotti, S. Merella, A. Capotondo, J. Sgualdino, G.Zanetti, C. von Kalle, M. Schmidt, L. Naldini, E. Montini, Lentiviral vector com-mon integration sites in preclinical models and a clinical trial reflect a benign in-tegration bias and not oncogenic selection, Blood 117 (2011) 5332–5339.

[108] M. Cavazzana-Calvo, E. Payen, O. Negre, G. Wang, K. Hehir, F. Fusil, J. Down, M.Denaro, T. Brady, K. Westerman, R. Cavallesco, B. Gillet-Legrand, L. Caccavelli,R. Sgarra, L. Maouche-Chretien, F. Bernaudin, R. Girot, R. Dorazio, G.J. Mulder,A. Polack, A. Bank, J. Soulier, J. Larghero, N. Kabbara, B. Dalle, B. Gourmel, G.Socie, S. Chretien, N. Cartier, P. Aubourg, A. Fischer, K. Cornetta, F. Galacteros,Y. Beuzard, E. Gluckman, F. Bushman, S. Hacein-Bey-Abina, P. Leboulch,


Transfusion independence and HMGA2 activation after gene therapy of humanbeta-thalassaemia, Nature 467 (2010) 318–322.

[109] D.A. Muruve, The innate immune response to adenovirus vectors, Hum. GeneTher. 15 (2004) 1157–1166.

[110] D. Descamps, K. Benihoud, Two key challenges for effective adenovirus-mediated liver gene therapy: innate immune responses and hepatocyte-specific transduction, Curr. Gene Ther. 9 (2009) 115–127.

[111] M.L. Batshaw, J.M. Wilson, S. Raper, M. Yudkoff, M.B. Robinson, Recombinant ad-enovirus gene transfer in adults with partial ornithine transcarbamylase defi-ciency (OTCD), Hum. Gene Ther. 10 (1999) 2419–2437.

[112] S. Lehrman, Virus treatment questioned after gene therapy death, Nature 401(1999) 517–518.

[113] H. Chen, Z.Q. Xiang, Y. Li, R.K. Kurupati, B. Jia, A. Bian, D.M. Zhou, N. Hutnick, S. Yuan,C. Gray, J. Serwanga, B. Auma, P. Kaleebu, X. Zhou, M.R. Betts, H.C. Ertl, Adenovirus-based vaccines: comparison of vectors from three species of adenoviridae, J. Virol.84 (2010) 10522–10532.

[114] A.P. McCaffrey, P. Fawcett, H. Nakai, R.L. McCaffrey, A. Ehrhardt, T.T. Pham, K.Pandey, H. Xu, S. Feuss, T.A. Storm, M.A. Kay, The host response to adenovirus,helper-dependent adenovirus, and adeno-associated virus in mouse liver, Mol.Ther. 16 (2008) 931–941.

[115] M.K. Chuah, D. Collen, T. VandenDriessche, Clinical gene transfer studies for he-mophilia A, Semin. Thromb. Hemost. 30 (2004) 249–256.

[116] J.R. Bischoff, D.H. Kirn, A. Williams, C. Heise, S. Horn, M. Muna, L. Ng, J.A. Nye, A.Sampson-Johannes, A. Fattaey, F. McCormick, An adenovirus mutant that repli-cates selectively in p53-deficient human tumor cells, Science 274 (1996)373–376.

[117] I. Ganly, D. Kirn, G. Eckhardt, G.I. Rodriguez, D.S. Soutar, R. Otto, A.G. Robertson,O. Park, M.L. Gulley, C. Heise, D.D. Von Hoff, S.B. Kaye, A phase I study of Onyx-015, an E1B attenuated adenovirus, administered intratumorally to patientswith recurrent head and neck cancer, Clin. Cancer Res. 6 (2000) 798–806.

[118] T.C. Liu, E. Galanis, D. Kirn, Clinical trial results with oncolytic virotherapy: a cen-tury of promise, a decade of progress, Nat. Clin. Pract. Oncol. 4 (2007) 101–117.

[119] F.R. Khuri, J. Nemunaitis, I. Ganly, J. Arseneau, I.F. Tannock, L. Romel, M. Gore, J.Ironside, R.H. MacDougall, C. Heise, B. Randlev, A.M. Gillenwater, P. Bruso, S.B.Kaye, W.K. Hong, D.H. Kirn, A controlled trial of intratumoral ONYX-015, aselectively-replicating adenovirus, in combination with cisplatin and 5-fluorouracil in patients with recurrent head and neck cancer, Nat. Med. 6(2000) 879–885.

[120] R. Cattaneo, T. Miest, E.V. Shashkova, M.A. Barry, Reprogrammed viruses as cancertherapeutics: targeted, armed and shielded, Nat. Rev. Microbiol. 6 (2008) 529–540.

[121] X. Li, Y.P. Zhang, H.S. Kim, K.H. Bae, K.M. Stantz, S.J. Lee, C. Jung, J.A. Jimenez, T.A.Gardner, M.H. Jeng, C. Kao, Gene therapy for prostate cancer by controlling ade-novirus E1a and E4 gene expression with PSES enhancer, Cancer Res. 65 (2005)1941–1951.

[122] E. Kim, J.H. Kim, H.Y. Shin, H. Lee, J.M. Yang, J. Kim, J.H. Sohn, H. Kim, C.O. Yun,Ad-mTERT-delta19, a conditional replication-competent adenovirus driven bythe human telomerase promoter, selectively replicates in and elicits cytopathiceffect in a cancer cell-specific manner, Hum. Gene Ther. 14 (2003) 1415–1428.

[123] R. Rodriguez, E.R. Schuur, H.Y. Lim, G.A. Henderson, J.W. Simons, D.R. Henderson,Prostate attenuated replication competent adenovirus (ARCA) CN706: a

selective cytotoxic for prostate-specific antigen-positive prostate cancer cells,Cancer Res. 57 (1997) 2559–2563.

[124] D.M. Nettelbeck, A.A. Rivera, C. Balague, R. Alemany, D.T. Curiel, Novel oncolyticadenoviruses targeted to melanoma: specific viral replication and cytolysis byexpression of E1A mutants from the tyrosinase enhancer/promoter, CancerRes. 62 (2002) 4663–4670.

[125] C. Conrad, C.R. Miller, Y. Ji, C. Gomez-Manzano, S. Bharara, J.S. McMurray, F.F.Lang, F. Wong, R. Sawaya, W.K. Yung, J. Fueyo, Delta24-hyCD adenovirus sup-presses glioma growth in vivo by combining oncolysis and chemosensitization,Cancer Gene Ther. 12 (2005) 284–294.

[126] J.W. Choi, J.S. Lee, S.W. Kim, C.O. Yun, Evolution of oncolytic adenovirus for can-cer treatment, Adv. Drug Deliv. Rev. (2011).

[127] J.J. Cody, J.T. Douglas, Armed replicating adenoviruses for cancer virotherapy,Cancer Gene Ther. 16 (2009) 473–488.

[128] H. Buning, S.A. Nicklin, L. Perabo, M. Hallek, A.H. Baker, AAV-based gene transfer,Curr. Opin. Mol. Ther. 5 (2003) 367–375.

[129] J.W. Bainbridge, R.R. Ali, Success in sight: the eyes have it! Ocular gene therapytrials for LCA look promising, Gene Ther. 15 (2008) 1191–1192.

[130] J.W. Bainbridge, A.J. Smith, S.S. Barker, S. Robbie, R. Henderson, K. Balaggan, A.Viswanathan, G.E. Holder, A. Stockman, N. Tyler, S. Petersen-Jones, S.S.Bhattacharya, A.J. Thrasher, F.W. Fitzke, B.J. Carter, G.S. Rubin, A.T. Moore, R.R.Ali, Effect of gene therapy on visual function in Leber's congenital amaurosis,N. Engl. J. Med. 358 (2008) 2231–2239.

[131] M. Giacca, A.H. Baker, Heartening results: the CUPID gene therapy trial for heartfailure, Mol. Ther. 19 (2011) 1181–1182.

[132] R.O. Snyder, C. Miao, L. Meuse, J. Tubb, B.A. Donahue, H.F. Lin, D.W. Stafford, S.Patel, A.R. Thompson, T. Nichols, M.S. Read, D.A. Bellinger, K.M. Brinkhous,M.A. Kay, Correction of hemophilia B in canine and murine models using recom-binant adeno-associated viral vectors, Nat. Med. 5 (1999) 64–70.

[133] M.A. Kay, C.S. Manno, M.V. Ragni, P.J. Larson, L.B. Couto, A. McClelland, B. Glader,A.J. Chew, S.J. Tai, R.W. Herzog, V. Arruda, F. Johnson, C. Scallan, E. Skarsgard, A.W.Flake, K.A. High, Evidence for gene transfer and expression of factor IX in haemo-philia B patients treated with an AAV vector, Nat. Genet. 24 (2000) 257–261.

[134] C.S. Manno, A.J. Chew, S. Hutchison, P.J. Larson, R.W. Herzog, V.R. Arruda, S.J. Tai,M.V. Ragni, A. Thompson, M. Ozelo, L.B. Couto, D.G. Leonard, F.A. Johnson, A.McClelland, C. Scallan, E. Skarsgard, A.W. Flake, M.A. Kay, K.A. High, B. Glader,AAV-mediated factor IX gene transfer to skeletal muscle in patients with severehemophilia B, Blood 101 (2003) 2963–2972.

[135] C.S. Manno, G.F. Pierce, V.R. Arruda, B. Glader, M. Ragni, J.J. Rasko, M.C. Ozelo, K.Hoots, P. Blatt, B. Konkle, M. Dake, R. Kaye, M. Razavi, A. Zajko, J. Zehnder, P.K.Rustagi, H. Nakai, A. Chew, D. Leonard, J.F. Wright, R.R. Lessard, J.M. Sommer, M.Tigges, D. Sabatino, A. Luk, H. Jiang, F. Mingozzi, L. Couto, H.C. Ertl, K.A. High,M.A. Kay, Successful transduction of liver inhemophilia byAAV-Factor IX and lim-itations imposed by the host immune response, Nat. Med. 12 (2006) 342–347.

[136] N.C. Hasbrouck, K.A. High, AAV-mediated gene transfer for the treatment of he-mophilia B: problems and prospects, Gene Ther. 15 (2008) 870–875.

[137] F. Mingozzi, K.A. High, Immune responses to AAV in clinical trials, Curr. GeneTher. 11 (2011) 321–330.


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