1. Introduction

2. Increasing the therapeutic

index

3. Cytoprotective agents

4. Gene therapy for

radioprotection

5. Adeno-associated viral

vectors (AAV)

6. Gene therapy with AAV

vectors

7. Gene therapy with retroviral

vectors

8. Radioprotective gene therapy

9. Conclusion

10. Expert opinion

Review

Radioprotective gene therapyPatrick Maier†, Marlon R Veldwijk & Frederik WenzHeidelberg University, University Medical Centre Mannheim, Department of Radiation Oncology,

Mannheim, Germany

Introduction: Radiation-induced myelosuppression or mucositis can limit the

effectiveness of radiotherapy by requiring dose reduction or delaying treat-

ment of tumour patients. The transfer of a radioprotective gene into normal

tissue cells would provide the opportunity to reduce the risks associated with

haematopoietic or intestinal toxicity after irradiation.

Areas covered: Several potentially radioprotective genes like multidrug resis-

tance 1 (MDR1), snail homolog 2 (SNAI2), and superoxide dismutases have

been evaluated in preclinical models for their radioprotective potential in

the last years. For gene transfer and ectopic expression, adenoviral, adeno-

associated virus (AAV) or retroviral vectors were used. The feasibility of radio-

protective gene therapy is discussed in consideration of the application of

cytoprotective agents and small-molecule protectors.

Expert opinion: Further vector optimization for targeted cell-specific

transduction and for more stable or regulated transgene expression is still

required. However, radioprotective gene therapy represents a very promising

method for reducing radiotherapy-related cytotoxicity of normal tissue cells

and thus may improve therapy success and the patient’s quality of life.

Keywords: AAV vectors, gene therapy, normal tissue cells, radioprotection, radiotherapy,

retroviral vectors

Expert Opin. Biol. Ther. (2011) 11(9):1135-1151

1. Introduction

Currently surgery, radiotherapy and chemotherapy represent the three clinically rel-evant therapy options in cancer treatment. About 60 -- 70% of all tumour patientsreceive radiotherapy. Combination of all three therapies in terms of a multimodaltreatment plan resulted in an increased cure rate during the last decades. It couldbe shown that local tumour control is of high relevance for the overall survival ofmany patients suffering from a variety of malignancies. However, for a furtherreduction in cancer-related mortality rates, additional improvements in therapeuticoptions are imperative. Furthermore, use of radiotherapy or chemotherapy affectsnot only the tumour cells but may result also in damage to normal tissue and aremainly causal for the haematopoietic syndrome (myelosuppression accompaniedby neutropenia or thrombocytopenia) and the gastrointestinal syndrome (mucositis,xerostomia, gastrointestinal bleeding and bacterial infections) [1-5]. Occurrence ofthese severe side effects often results in a dose reduction or in a delay or even termi-nation of the radio- (chemo-) therapy. Since tumour control probability primarilycorrelates with the dose, the occurrence of such events would significantly impedetherapy success.

2. Increasing the therapeutic index

Normal tissue reactions and tumour control (and thereby the success probability ofuncomplicated cure by radio- or chemotherapy) are both dependent on the applieddose. The rate of normal tissue damage must always be set in relation to the rate oftumour control [6] defining the therapeutic index or therapeutic ratio. Thus,

10.1517/14712598.2011.580271 © 2011 Informa UK, Ltd. ISSN 1471-2598 1135All rights reserved: reproduction in whole or in part not permitted

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broadening this ratio, the complication-free interval betweentumour control and therapy-related normal tissue complica-tions, is of great interest in both radiotherapy and chemother-apy (Figure 1). Ideally, a therapy should result in a high aspossible tumour control probability, yet should keep the nor-mal tissue complication probability as low as possible [6]. Forradiotherapy, this would ideally mean that tumour controlcan be achieved at significantly lower irradiation doses thanrelevant normal tissue damage.An advancement to increase the therapeutic index and

thereby tumour control, has been the use of precise,computer-controlled beam application in combination withmodern imaging techniques and three-dimensional treatmentplanning as used in intensity modulated radiotherapy (IMRT)or image guided radiotherapy (IGRT) [7-9]. Stereotactic irradi-ation techniques yield therapeutic gain by reducing thedose applied to the normal tissue and by conforming the iso-dose to the target volume. Thus, more exact definition of thetumour volume and reduction of the margins required aroundthe target volume allow escalation in dose delivered to thetumour volume with the potential for increased cure rates.An additional way to increase the therapeutic index by

reducing normal tissue damage, especially late effects likefibrosis, pneumonitis or myelopathy could be achieved bythe use of fractionated irradiation. Here advantage is takenof the higher recovery rate between two radiation fractionsof normal tissue cells compared with tumour cells.

Another option to prevent normal tissue from damage, isthe use of radiosensitizing substances that could increase theresponse of tumour cells to radiotherapy like inhibitors fortopoisomerase I (topotecan/irinotecan or camptothecin andderivatives), poly(ADP-ribose) polymerase, histone deacety-lase or heat shock protein 90 [10]. All these substances muststill be evaluated in clinical trials. Thus, further concepts arewelcome to reduce normal tissue damage.

3. Cytoprotective agents

Substances for radioprotection of normal tissue have been sub-ject of intense research for more than 60 years [11]. Thisresearch was primarily motivated to treat victims of a nuclearattack, yet since the 1980s normal tissue protection duringradiotherapy has been propelled into the focus [11]. Thus,some of these substances have already been tested in preclinicalor clinical studies during radiotherapy (Table 1) [12].

Amifostine (WR-7221), a thiol derivative, was originallydeveloped for radioprotection during a nuclear war. It isadministered as a prodrug, which gets activated by a cellu-lar-membrane-localized alkaline phosphatase and accumulatesprimarily in salivary glands, kidneys and intestinal mucosa.Amifostine mainly acts as a scavenger of free radicals, whichare generated during radiotherapy [13]. Amifostine has alreadybeen tested for normal tissue protection during radiotherapyof squamous cell carcinoma of the head and neck, NSCLCand pelvic malignancies. In all these studies, a radioprotectiveeffect in tumour tissues could not be shown. Concomitantly,the risk for mucositis, esophagitis, xerostomia, dysphagia,acute pneumonitis and cystitis was reduced [14,15]. However,the overall response rates for patients with head and neck can-cers, NSCLC or pelvic malignancies did not differ betweenamifostine and control groups. Currently, amifostine is onlyapproved in the USA and in Europe to inhibit developmentof xerostomia in patients with head and neck cancer afterradiotherapy alone. For the use of amifostine for othertumour entities conclusive results are still pending.

Another potentially radioprotective substance is palifermin,a recombinant protein of keratinocyte growth factor 1(KGF1) [16]. Palifermin stimulates proliferation, migrationand differentiation of epithelial cells and is also involved inDNA repair and protection against oxygen radicals [17]. Pro-tection of the gut mucosa by palifermin could be shown inpreclinical studies which was not accompanied by a negativeeffect on the benefit of 5-fluorouracil in combination withradiotherapy [18]. Otherwise, an enhanced kill of tumour cellsof gastric and breast cancers was detectable. The results ofclinical trials for the use of palifermin for treatment of squa-mous cell carcinoma of the head and neck are still limitedaccording to the safety and efficacy of palifermin [12].

Several other factors, like recombinant human erythro-poetin (rhEPO), G-CSF, and GM-CSF, have been evaluatedin clinical trials. However, the results advise against the useof these substances for radioprotection. Application of rhEPO

Article highlights.

. Gene-therapy-based normal tissue radioprotection ismore promising than tumour radiosensitization sincesignificantly lower amounts of successfully transducedcells are needed to obtain clinically relevant effects.

. Lentiviral transfer of multidrug resistance 1 (MDR1) tohuman haematopoietic stem cells results in significantradioprotection after single-dose andfractionated irradiation.

. Using lentiviral overexpression of snail homolog 2(SNAI2), proof of principle for radioprotection could beshown in a lymphoblastoid cell line.

. Viral and non-viral gene therapeutic overexpression ofmanganese superoxide dismutase (MnSOD) was shownto be radioprotective in a variety of normal tissue cells.Interestingly, it also resulted in a lack of radioprotectionin tumour cells.

. Improved design of viral vectors can lead to targetedcell-specific transduction and to increased stable or toradiation-controlled transgene expression, therebyfurther improving the feasibility of a future selectivein vivo radioprotective gene therapy application.

. Gene therapy-based radioprotection of normal tissuecells has the potential to become an additional approachto increase the therapeutic index in radiotherapy andthereby potentially improve therapy success and thepatient’s quality of life.

This box summarizes key points contained in the article.

Radioprotective gene therapy

1136 Expert Opin. Biol. Ther. (2011) 11(9)

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resulted in patients with radiotherapy in an increased risk ofthromboembolic effects and in a possible stimulation oftumour growth [19,20]. Use of G-CSF or GM-CSF was not fol-lowed by a reduction of the amount of mucositis but on thecontrary resulted in enhanced side effects like myelotoxicityor thrombosis [21,22].

In the last two years several small molecule protectorslike Gramicidin-S-derived-nitroxide, triphenylphosphonium-conjugated nitroxides, glyburide (a clinically used hypoglyce-mic drug), and inhibitors of glycogen synthase kinase 3b(SB216763 and SB415286) were identified and characterizedin preclinical studies [23-26]. Additionally, two classes of anti-biotics, tetracyclines and fluoroquinolones, were shown tobe radioprotective in mice [27]. The radioprotective potentialof all these compounds has to be evaluated in clinical settings,especially to ensure that systemic application causes radio-protection exclusively in normal tissue cells and not also intumour cells.

4. Gene therapy for radioprotection

As an alternative approach for systemic administration ofsmall-molecule protectors, gene therapy is a very promisingmethod for protection of normal tissue cells during radio-or chemotherapy of tumour diseases [28,29]. Inserting a specificDNA can replace a defective gene or deliver the informationfor an additional gene (e.g., a radioprotective gene) whoseexpression is necessary for the treatment of a disease. For anin vivo gene therapy the therapeutic vector will be systemicallyapplied, for an ex vivo approach the target cells likehematopoietic stem cells (HSC) will be gained from the

patient, modified by gene therapy in the culture dish andretransplanted subsequently. Thus, the modus operandi fora protective gene therapy involves the modification of healthycells by a gene therapy vector encoding a protein delivering aprotection against irradiation damage.

Either non-viral or viral vectors are being used as vector sys-tems for gene transfer [30]. Non-viral vector systems (directnaked DNA injection, cationic liposomes or DNA--polymerconjugates) have three main advantages compared with viralvector systems: they do not activate the inflammatory andimmune response of the host [31], are relatively inexpensive,and large fragments of DNA can be transferred. However,the disadvantages prevail; non-viral vector systems are ofteninefficient and primarily result in transient gene transfer [32].

On the other hand, generally higher transduction efficien-cies can be achieved with viral vector systems [33] since duringevolution viruses have adopted very efficient mechanisms tointroduce their genomes into their target cell and to expresstheir genes subsequently. Yet viral vector systems are generallymore cumbersome to produce and more expensive. In fact,the advantages outweigh these disadvantages, as viral vectorshave been used in about 75% of the hitherto nearly 1650 clin-ical gene therapy trials [34]. This review focuses on two viralvector systems: adeno-associated-virus- and retrovirus-basedvectors (for information on additional vector systems see [33]).

5. Adeno-associated viral vectors (AAV)

Wild type AAV was first discovered as a contaminant ofadenoviral samples in the 1960s and was later also detectedin humans [35]. The colocalisation with adenoviral particlesis not unexpected, as the virus requires several gene productsfrom a helper virus (e.g., adeno- or herpesvirus) for efficientreplication [36,37]. AAV is a non-enveloped, replication-deficient virus belonging to the parvoviridae and comprisesa single-stranded DNA genome [38] of 4.7 kb and is flankedby the inverted terminal repeats (ITRs; Figure 2). The ITRsfunction as origins of replication and contain the packagingsignal [38,39]. The ITRs are the only wild-type AAV sequencesrequired in cis (must be on the viral genome) to allow produc-tion of replication-deficient recombinant AAV (rAAV) par-ticles (Figure 3A), whereas the other AAV elements can bedelivered in trans.

The wild type AAV genome contains three open readingframes (ORFs): the first encodes the four non-structural Repproteins that control and regulate AAV replication [40],whereas the second encodes the three viral capsid proteins(Figure 2) [41,42]. Recently, an alternative ORF was detectedencoding the ‘assembly-activating protein’ (AAP) [43].

The tropism of AAV is determined by the binding sites onthe capsid proteins and is therefore serotype-dependent.A variety of serotypes have been isolated from primates, ofwhich three have been detected in humans (AAV2, 3and 5). AAV2 has been most extensively investigated andhas therefore been primarily used in AAV-based gene transfer

0

20

40

60

80

100

0 20 40 60 80

NTCP

Uncomplicatedcure

Tumoursensibilization

Fractionatedradiotherapy

Normal tissueprotection

Conformalradiotherapy

[%]

Dose [Gy]

TCP

TI

Figure 1. Dose-response relationship between tumour

control propability (TCP) and normal tissue complication

propability (NTCP) during radiotherapy. The therapeutic

index (TI) is the area between the TCP and NTCP curves.

Sensitization of the tumour cells for radiotherapy would

result in a shift of the TCP curve towards lower doses,

whereas radioprotection of normal tissue cells would result

in a shift of the the NTCP curve towards a higher doses. Each

of these approaches would result in an increased

therapeutic index.Modified from [6].

Maier, Veldwijk & Wenz

Expert Opin. Biol. Ther. (2011) 11(9) 1137

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studies. Due to differences in tropism and immunogenicity,recombinant vector systems based on novel serotypes havebeen developed. For example: AAV8 and AAV7 vectors areable to transduce hepatocytes and murine muscle cells withhigher efficiency than AAV2, respectively [44]. AAV6-basedvectors share many features of AAV1 and AAV2, yet are lessimmunogenic in humans [45].Overall, AAV-based vectors exhibit several beneficial fea-

tures for gene therapy, like the lack of association with anydisease. In contrast, wild type AAV has been credited withoncoprotective properties [46-48]. In addition, AAV-based vec-tors are able to transduce non-dividing cells and AAV particlesare highly stable, allowing purification and showing increasedhalf life in comparison with other vectors. Also the low immu-nogenicity compared with other vectors is highly beneficial, asespecially after the first gene-therapy-related death [49], immu-nogenicity and safety are more than before of great concern inclinical studies. The use of other non-human serotypes willimprove these even further.AAV vectors also have some disadvantages; its genome is

relatively small (5 kb), thereby restricting transgene size.Unlike the wild type, recombinant AAV is not able to inte-grate specifically on chromosome 19q13.4, as they lack theessential Rep proteins and promoter region [50,51].For above mentioned reasons, rAAV vectors are most suit-

able for either short-term expression in dividing tissues (‘hit-and-run’) or long-term gene transfer into post-mitotic/slowly dividing tissues, for example neurons or muscle tis-sue [44,52]. Due to their low rate of integration, rAAV vectorshave a minute chance of insertional mutagenesis comparedto retroviral vectors.

6. Gene therapy with AAV vectors

Due to their favourable characteristics, recombinant AAVvectors have been used in a variety of preclinical and clinical

studies. Currently 75 clinical studies with these vectors havebeen performed or are still ongoing [34].

Promising results based on therapeutic effect have beenobtained for AAV-based clinical studies for the treatment ofmonogenetic diseases. These comprise 39% for AAV, whereasthe percentage for all vectors is only 8%, thereby clearlyfavouring AAV-based vectors for this indication. Moststudies in this group are either for the treatment of cysticfibrosis [53,54] or coagulation deficiencies [55,56]. The targetedcells favour AAV (respiratory epithelium and hepatocytes,respectively), as these are well susceptible to AAV2-basedvectors. Yet although studies in large-animal models andnon-human primates were very promising (e.g. factor IX indogs) [57], no significant clinical benefit was observed. For fac-tor IX, this was primarily based on the lack of gene transferefficiency in humans due to pre-existing immunity to theAAV2 vectors [56]. Still, these studies are highly promising,as a switch of vectors to a different AAV serotype or library-selected mutant vectors [58] where no pre-existing immunityis to be expected in humans, may finally pave the way forclinical benefit.

Recently highly promising results have been obtained inAAV-based gene transfer studies for the treatment of neuro-logical and ocular diseases, currently accounting for 19 and8% of all AAV-based clinical studies, respectively. Followingsuccessful experiments in non-human primates [59], AAV2-based vectors have been employed in several studies for treat-ing Parkinson’s disease, where significant gene transfer anddetectable clinical success could be shown [60,61].

After highly promising results were obtained in a dogmodel for Leber’s congenital amaurosis, a disease causingloss of sight [62,63], a real breakthrough was presented in2008 as several groups presented data showing efficient genetransfer with AAV2-based vectors into the human retinalepithelium and improved vision [64,65], even after over ayear [66,67].

Table 1. Cytoprotective agents combined with radiotherapy.

Radioprotective

agent

Substance type Normal tissue effects Tumour effects Clinical appoval

Amifostine Thiol derivative Reduced mucositis, esophagitis,xerostomia, dysphagia, acutepneumonitis and cystitis

No tumour protection Approved in USA andEurope to inhibit xerostomiain patients with head andneck cancer after radiotherapyalone

Palifermin recombinant KGF1 Protection of gut mucosa inpreclinical models

No tumour protectionin preclinical models

Still under clinical evaluation

G-CSF/GM-CSF Recombinant proteins No reduced mucositis butenhanced side effects likemyelotoxicity or thrombosis

nd Not approved forradioprotection

rhEPO Recombinant protein Increased risk ofthromboembolic effects

Possible stimulation oftumour growth

Not approved forradioprotection

KGF1: Keratinocyte growth factor 1; nd: not determined; rhEPO: Recombinant human erythropoetin.

Radioprotective gene therapy

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In summary, for a variety of indications, the future ofAAV-based clinical gene transfer seems highly promising.

7. Gene therapy with retroviral vectors

Retroviral vectors are a complete different type of viral vectorscompared to AAV vectors. After infection, the genome con-sisting of two copies of single-stranded RNA molecules of7 -- 12 kb is reverse-transcribed into double-stranded cDNAand integrated into the genome of the host cell. Most gam-maretroviral vectors were developed on the basis of theMoloney murine leukaemia virus (MoMLV) [68]. In recentyears, lentiviral vectors primarily based on HIV have beenincreasingly used for gene transfer [69]. Their biggest advan-tage compared with gammaretroviral vectors is their abilityto infect quiescent cells [70]. The scheme of a typical lentiviralvector which has actually been used in clinical trials isshown in Figure 3B. Detailed information about all aspectsof retroviral vectors can be found elsewhere [71].

Since a first application in a clinical trial 20 years ago, up tonow retroviral (gammaretroviral and lentiviral) vectors havebeen used in more than 350 gene therapy studies. Retroviralvectors are particularly suited for gene-correction of cells dueto long-term and stable expression of the transferred trans-gene(s) and also to the little effort required for their cloningand production. Several monogenic inherited diseases, mostlyimmunodeficiencies, have been successfully treated in recentyears [72]. Especially the successful gene therapy trials for

X-linked adrenoleukodystrophy (ALD) [73] and for adenosinedeaminase deficiency resulting in severe combined immuno-deficiency (ADA-SCID) [74] justified the statement ‘GeneTherapy Returns’ in the section breakthrough of the year inthe Science issue of 18th December 2009. Already in January2009 Kohn and Candotti entitled their editorial in TheNew England Journal of Medicine ‘Gene Therapy FulfillingIts Promise’ [75]. Nevertheless it must be mentioned thatretroviral vectors were correlated with negative outcome ofgene therapy studies. After gene therapy with a gammaretro-viral vector, 5 out of 20 children with X-linked SCID(SCID-X1) developed a T-cell acute lymphocytic leukaemia(T-ALL) within 2 -- 5 years, 1 of these children died [76]. Suc-cessful chemotherapy resulted in a restoration of polyclonaltransduced T cell populations for three children with contin-ued benefit from the therapeutic gene transfer. The occur-rence of T-ALL was attributed to the so-called insertionalmutagenesis, which means that expression of genes in thevicinity of the integrated provirus was upregulated due tothe strong viral long terminal repeat (LTR)-located promoter.However, further analyses of the leukaemic clones of these fivechildren revealed further genetic abnormalities [77,78]. Thus, amulti-step model with the insertional mutagenesis as oneof several steps must be proposed for the development ofthese leukaemias.

HSC were the target cells for ex vivo modification by theretroviral vector in all above mentioned successful gene ther-apy studies. A reduction of myelotoxicity is the focus of aradioprotective gene therapy of HSC. Due to their pluripo-tency HSC and their differentiated descendants expressthe transgene after gene therapeutic modification with anintegrating retroviral vector. HSC can be easily mobilizedfrom their niches in the bone marrow by application ofG-CSF [79] or Plerixafor [80] and subsequently isolated bymagnetic-immunobeads-labelled antibody against the surfacemolecule CD34 [81], which is expressed by these progenitorcells [82]. An ex vivo gene transfer of radioprotective genesto the patient’s HSC is followed by their retransplantationallowing subsequent application of irradiation fractionswith higher intensities accompanied by reduced myelotoxicside-effects and increased effect on the tumour cells [83].Until now a few genes have been evaluated in in vitro andpartially in vivo (small animal) radioprotective gene transferstudies (Table 2).

8. Radioprotective gene therapy

8.1 MDR1MDR1 (multidrug resistance 1) encodes a 170 kDa trans-membrane protein and is a member of the ATP-bindingcassette superfamily (therefore p-glycoprotein (PGY1) orABCB1 are alternative gene names) [84]. MDR1 has an estab-lished role as a mediator of cytotoxic drug resistance [85]

pumping hydrophobic cytostatics like doxorubicin, etoposide,paclitaxel and vincristine out of the cell. Thus, cells expressing

p5 p19 p40

Rep78 4.2 kb

Rep68 3.9 kb

Rep52 3.6 kb

Rep40 3.3 kb

VP1 87 kDa 2.6 kb

VP2 73 kDa 2.3 kb

VP3 62 kDa 2.3 kb

AAV2ITR ITR

pA

Rep Cap

AAP 23 kDa 1.0 kb

Figure 2. The wild type adeno-associated virus

type 2 (AAV2) genome, open reading frames and splice

products. The AAV2 genome is representatively depicted, as

all serotypes share a common structure with high sequence

homology. The two open reading frames (replication (Rep)

and capsid (Cap)) are depicted as gray boxes, the untrans-

lated regions and splice sites as straight and zigzag lines,

respectively.Modified from: Kotin [159] and Sonntag et al. [43].

ITR: Inverted terminal repeats; pA: Poly adenylation signal.

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MDR1 exhibit increased resistance against the cytotoxic effectof these substances. MDR1 is not only highly expressed intumour cells but also in various normal tissue cells (e.g., liver,kidney and endothelial cells of the blood--brain-barrier) [86].Also HSC express MDR1 but lose its expression duringdifferentiation [87]. Thus, MDR1 gene therapy of HSC wouldresult in a protection of differentiated haematopoieticcells. MDR1 gene transfer has already been established forchemoprotection of HSC in the last two decades and usedin clinical trials for bone marrow protection in patients under-going high-dose chemotherapy and autologous stem-celltransplantation [88,89].However, MDR1 mediates not only chemoprotection

by drug efflux but has also been found to inhibit apoptosisinduced by chemotherapeutics, death receptor ligands, serum

starvation and UV or ionizing irradiation [90,91]. Both anefflux-dependent mechanism by inhibition of FAS-inducedactivation of caspase-8 [90] and an efflux-independent mecha-nism [91] have been described. Thus, this antiapoptotic effectafter irradiation qualifies MDR1 as a potential gene forradioprotection of normal tissue cells.

In a first approach to assess its safety, we were interested ifMDR1-overexpression influences the expression of othergenes. We could show by micorarray analysis that retroviraloverexpression of MDR1 in the human B cell lymphoblastoidTK6 cell line resulted in significantly differential expressionof 61 annotated genes (31 upregulated, 30 downregulatedwith p < 10-4) [92]. Several genes coding for proteins involvedin exocytosis or detoxification were significantly upregulatedin TK6MDR1 cells. Additionally, proapoptotic genes

Promoter Transgene

3’ LTR 5’ LTR cPPTRRE WPRE

A.

B.Marker gene

Linker

Ψ

ITR ITR

SD/SA pA pA

PEF1a pTK

Gene 1 Gene 2

Figure 3. Structures of recombinant viral vectors. A. A recombinant AAV vector. As only the ITRs are required, the rest of the

AAV genome can be replaced with the transgene(s) and regulatory elements. Here an example vector is depicted (based on

pTR-UF5 [160]), containing two promoters (the elongation factor 1a (EF1a) and thymidine kinase (TK) promoters) driving the

expression of the two transgenes (e.g.,: CuZnSOD) and each terminated by an poly adenylation signal (pA). SD/SA; splice

donor/splice acceptor. B. A recombinant HIV-based lentiviral vector contains of the original HIV genome only the long

terminal repeat (LTR) sequences and the packaging signal Y. The transgene which can be linked by either an internal

ribosome entry site (IRES) or a 2A-sequence with a marker or selection gene in a bicistronic expression cassette is controlled by

an internal promoter. Rev-responsive element (RRE) is the binding region of the viral regulator of expression of the virion

(REV) protein, which is encoded on the packaging plasmid, thus enabling the nucleocytoplasmic transport of viral RNA

required for the translation in the cytoplasm. The central polypurine tract (cPPT), a cis-active sequence supports the

translocation of the reverse-transcribed viral cDNA into the nucleus and the woodchuck hepatitis virus posttranscriptional

regulatory element (WPRE) enhances the stability of the RNA resulting in increased transgene expression [71].

Table 2. Candidate genes for radioprotective gene therapy.

Gene Protein function Used transfer vector Radioprotective effects

MDR1 Efflux pump, inhibitionof caspase activity

Gammaretro-/lentivirus Radioprotection and reduced apoptosis of TK6 cells,radioprotection of human HSC

SNAI2 Transcription factor,inhibtion of transcriptionof proapototic PUMA

Lentivirus Radioprotection and reduced apoptosis of TK6 cells

SOD1/SOD2 Detoxifies superoxideradicals

Plasmid liposomes,AAV, adenovirus and lentivirus

Radioprotection in a variety of normal tissues(murine and human)

AAV: Adeno-associated virus; HSC: Hematopoietic stem cells; MDR1: Multidrug resistance 1; PUMA: p53 upregulated modulator of apoptosis; SNAI2: Snail

homolog 2; SOD: Superoxide dismutase.

Radioprotective gene therapy

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(caspase1 (CASP1), CASP4 and NACHT domain leucine-rich repeat and pyrin domain containing 7 (NALP7)) weredownregulated with concomitant increased expression of thepotentially antiapoptotic gene v-akt murine thymoma viraloncogene homolog 3 (AKT3). In functional assays the influ-ence of MDR1 overexpression on apoptotic signaling wasfurther corroborated by reduced apoptosis rates in responseto irradiation in TK6MDR1 cells. Colony formation assaysshowed that TK6MDR1 cells were significantly less radiosen-sitive in the dose range 1 -- 4 Gy compared with untransducedTK6 or TK6neo (containing neomycin phosphotransferaseas transgene) cells. The surviving fraction at the clinically rel-evant dose of 2 Gy, SF2, was 1.7-fold higher in TK6MDR1relative to untransduced TK6 cells.

In further studies we evaluated the radioprotective potentialof MDR1 in primary human HSC [93]. For this reasonMDR1 was cloned into a lentiviral self-inactivating (SIN) vec-tor. Transduced human undifferentiated HSC were irradiatedwith 0 -- 8 Gy, held in liquid culture under myeloid-specificmaturation conditions, and after 12 days, the amounts ofMDR1-expressing cells were determined by a functional assaytesting for the export of the MDR1-substrate Rhodamine [94].The proportion of MDR1-positive cells of four humandonors increased with increasing radiation doses (up to14-fold increase at 8 Gy). Determination of expression ofmyeloid-specific surface marker proteins revealed that mye-loid differentiation was not affected by transduction andMDR1-overexpression. Furthermore, irradiation of myeloiddifferentiated cells showed an increase of MDR1-positive cellswith escalating radiation doses (e.g., 12.5 -- 16% from0 -- 8 Gy) demonstrating a radioprotective effect of MDR1.Most importantly, fractionated irradiation (3 � 2 Gy at24 h intervals) of MDR1-transduced TK6 cells and of humanHSC resulted in an increase in MDR1-positive cells (Figure 4).The same fractionation yielded also in an increase of the sur-viving fraction of TK6MDR1 cells relative to untransducedTK6 cells. Thus, our in vitro data clearly showed a protectiveeffect of retroviral overexpression of MDR1 increasing theradiotolerance of haematopoietic cells.

In a next step the radioprotective effect were verified in theNOD/SCID mouse xenotransplantation model [94] withtransplanted human HSC which were transduced with thelentiviral MDR1-overexpressing vector (unpublished results).Before transplantation all mice were myeloablatively irradi-ated with 3 Gy. After an engraftment period of 6 weeksmice were irradiated with a radiotherapeutic dose of 1.5 Gy,3 and 14 days later bone marrow, spleen and peripheral bloodwere analyzed for human and MDR1-expressing cells. Thetransduction rate of the HSC with the lentiviral MDR1-vector was up to 21% as determined in parallel cell culture.However, no MDR1-overexpressing cell could be determinedat the time points of analysis after radiotherapy. In contrast,by a quantitative real-time PCR specific for the viral vectora significant proportion of transduced cells could be detected.This result suggested that the transgene expression was

silenced by variegation effects and highlighted the necessityfor a modified structure of the lentiviral vector in order toachieve a long-term stable transgene expression in vivo.

8.2 SNAI2Another very promising gene for radioprotection is snailhomolog 2 (SNAI2) which belongs, together with SNAI1and 3, to the evolutionary conserved SNAIL family of zincfinger transcription factors [95]. The important role ofSNAI2 for survival of HSC after irradiation was shown withSNAI2-/- mice which were more radiosensitive than wild-type mice, showing a significant loss of haematopoietic cellsby apoptosis and increased mortality after irradiation [96].Transcription of SNAI2 is induced by p53 after irradiation;on the other hand SNAI2 inhibits p53-induced transcrip-tion of p53 upregulated modulator of apoptosis (PUMA)(Figure 5). PUMA expression in response to irradiation wasshown in HSC and in intestinal crypt cells [97,98]. PUMA-deficient mice had blocked apoptosis in the intestinal progen-itor and stem cells, showed enhanced crypt proliferation andregeneration, and prolonged survival following lethal dosesof radiation [97]. PUMA activates radiation-induced apoptosisvia the mitochondrial pathway by inhibition of B celllymphoma 2-like 1 (BCLXL) and activation of B-cell lym-phoma 2-associated X protein BAX [99]. Development ofsmall-molecule PUMA inhibitors for protection againstPUMA-dependent and radiation-induced apoptosis byinhibition of the interaction between PUMA and BCLXLis already under investigation [100]. Thus, suppression ofPUMA expression by gene therapy with the transcriptionrepressor SNAI2 as a transgene might be an approach for nor-mal tissue protection during radiotherapy. Delivery of a viralvector encoding SNAI2 to HSC or to gastrointestinal tissuesmight protect these cells from radiation-induced damage. Inorder to evaluate the potential of SNAI2 as a radioprotectorwe cloned the corresponding cDNA into the lentiviral SINvector in a bicistronic expression cassette with enhanced greenfluorescent protein (EGFP) as marker gene and an internalribosome entry site (IRES) sequence as a linker [101]. As thecellular model system again TK6 cells were used; radiation-induced apoptosis in TK6 cells is known to be mediated bythe p53-dependent mitochondrial pathway [102,103]. Wedetected an up to fourfold increase in PUMA transcription2 -- 8 h after irradiation of TK6 cells with 2 Gy (16 h laterthe expression nearly returned to the base level). Due to thisincrease of PUMA expression in response to irradiation theTK6 cell line is a good functional model to assay the radiopro-tective effect of SNAI2 overexpression. SNAI2 overexpressionresulted in a significant radioprotective effect in a cytotoxicityassay after irradiation with 0 -- 5 Gy as compared withuntransduced or control vector (inverse-oriented SNAI2cDNA)-transduced cells. Determination of the proportionof apoptotic cells in SNAI2-overexpressing cells revealed a sig-nificant reduction in apoptosis as compared with both controlentities. Additionally, SNAI2-overexpressing cells showed a

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survival advantage in a clonogenic assay after irradiation with0 -- 3 Gy as compared with the control-vector-transducedcells. Therefore, in this study we could provide proof of prin-ciple that lentiviral overexpression of SNAI2 might be usedfor radioprotective gene therapy in order to widen the thera-peutic range in radiotherapy. Validation in human primarycells is in progress.

8.3 Superoxide dismutaseSuperoxide dismutase (SOD) has been used in several radio-protective gene transfer studies. SOD catalyses the dismuta-tion of superoxide into hydrogen peroxide and oxygen andis of high relevance in the defence against superoxide radicals.

4 O H H O O2 2 2 2•− ++ → +4 2 2

In humans, three different enzymes belonging to this class areknown: CuZnSOD (SOD1) [104,105], MnSOD (SOD2) [106]

and extracellular CuZnSOD (SOD3) [107]. Whereas CuZn-SOD resides as a 32 kDa homodimer in the cytoplasm ofthe cell with each unit containing one molecule of Cu andZn, MnSOD (96 kDa; 4 � Mn) is located in the mitochon-dria and SOD3 (130 kDa; 4 � Cu, 4 � Zn) extracellularly;both the latter proteins are tetramers. In other organisms, sim-ilar enzymes are known, in which the metal ion is replaced byFe or Ni.

As during irradiation, beside the direct damage to theDNA, also reactive oxygen species (ROS) are generated byradiolysis that can indirectly damage the DNA, the detoxifica-tion of these can potentially positively contribute to thesurvival of a cell during irradiation. Although the exact pro-portion and relevance of superoxide radicals after irradiationof cells is not known, it has been shown that cells react toirradiation with an upregulation of MnSOD, hinting at a

correlation between cellular radiation and superoxide radicalformation [108,109]. Although the latter radical may not be asreactive as the hydroxyl radical, the prime product after ioniz-ing irradiation of cells, it could be shown that in a cell thesuperoxide radical can be converted into more reactivemetabolites such as hydroxyl or peroxynitrite radicals [110].

In addition to being able to detoxify the superoxide radicalanion, CuZnSOD has been shown to exhibit an antifibroticeffect by TGF-b1 repression and reversion of myofibroblastsinto normal fibroblasts [111]. MnSOD was shown to suppressapoptosis by several routes: stabilization of the mitochondrialmembrane [112], suppression of cytochrome c and caspase9 release [113] and MnSOD-induced downregulation ofp53-mediated apoptosis [114].

Due to their above mentioned properties, several groupshave determined the radioprotective effects of the SOD genes(especially MnSOD) with the goal of radioprotective genetherapy. For CuZnSOD, a radioprotective effect is still con-troversial. Epperly et al. [115] did not observe any protectiveeffect of CuZnSOD after adenoviral overexpression in amouse model. Similarly, Sun et al. [116] could not detectincreased radioresistance of CuZnSOD after transfectioninto CHO cells. Interestingly, Delanian et al. [117] were ableto obtain a reduction in radiation-induced apoptosis afteraddition of CuZnSOD-filled liposomes to human fibroblasts.That the observed discrepancies were not caused by differen-ces in the direct application of protein versus gene transfercould be shown by our group [118]: again in human fibro-blasts, radioprotection was observed, although this time aftergene transfer of the CuZnSOD gene with a recombinantAAV2-based vector.

Also for MnSOD, the radioprotective effect is notunivocally clear. Whereas some groups were able to showradioprotection of cells after MnSOD overexpression, others

0

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C.TK6TK6MDR1

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Figure 4. Radioprotection of TK6 cells or human hematopoietic stem cells (HSC) by multidrug resistance 1 (MDR1)

overexpression. Fractionated irradiation of MDR1-transduced TK6 or HSC (from three different donors) resulted in an

increase in MDR1-positive cells (rhodamine-exporting cells). (A.) TK6 cells were transduced with the MDR1 vector with a

multiplicity of infection (MOI; viral particles per cell) of 1 or 10. (B.) HSC cells were transduced with a MOI of 10. Cells were

irradiated up to three times with fractions of 2 Gy. Proportions of MDR1-positive TK6 cells (A.) or HSC (B.) or survival fractions

of T6 cell (C.) were determined.Reproduced from [93] with permission from Radiation Research.

Radioprotective gene therapy

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did not detect any. Using a MnSOD-gene-containing plas-mid/liposome transient transfection system in a murine mousemodel, the group of Greenberger and colleagues showed adecrease in irradiation-induced acute and chronic damage, aswell as a reduction in lung fibrosis and organizing alveolitisafter irradiation [119,120]. Similar results were observed usingSOD-overexpressing adenoviral vectors in the samemodel [115].The MnSOD-plasmid/liposome constructs were also success-fully used in the prevention of irradiation-induced esophagitisin a murine mouse model [120,121] and organ explant cul-tures [122,123]. In addition, MnSOD-mediated radioprotectioncould be shown in the murine bladder [124], oral cavity [125],and in cryopreserved HSC from the bone marrow [126]. Results

from our group could also show radioprotection by MnSODafter AAV2-based gene transfer into primary human lungfibroblasts [118].

Yet on the other hand, no significant differences in radio-sensitivity was observed after overexpression of either CuZn-SOD or MnSOD in a cervix carcinoma cell line (HeLa) [127]

or a lymphoblastoid cell line (TK6 (unpublished results)using AAV- and lentiviral-based vectors, respectively). Forthe TK6 cells, even despite a MnSOD-induced reduction inradiation-invoked apoptosis, this had no relevance to the clo-nogenic survival after irradiation. It should be noted that inboth setups significant overexpression of the respective vectorsand transgene, as well as SOD activity could be obtained.Interestingly, also others observed a lack of radioprotection byMnSOD using gene-transfer-mediated overexpression. Zhonget al. [128] observed this in a rat glioma cell line under in vitroconditions, whereas the Greenberger group obtained similardata transplanting a rat lung carcinoma cell line into a mousemodel [119,125]. All the cells where MnSOD overexpression didnot result in radioprotection share a feature that may be thebasis of the observed effect: where non-malignant cells couldbe protected by MnSOD, all insensitive cells were derivedfrom malignant cells/cell lines. Our group could clearlyshow this using the same vector system (AAV2-based) onboth tumour (HeLa) and normal tissue cells (primary lungfibroblast), thereby eliminating other variables beside thecells [118,127]. Epperly et al. showed similar result in a murinetest model: the normal lung tissue was protected, yet the oth-otopic mouse tumour cells were not after application of theirMnSOD-plasmid/liposomes and subsequent irradiation [119].Recently, Borrelli et al. also showed radioprotection in normaltissue cells and radiosensitisation in tumour cells, afterapplication of recombinant MnSOD to the lungs of miceand subsequent irradiation [129].

There are several hypotheses as to why MnSOD maybehave so paradoxically in the two cell entities. One was sug-gested by Zhong and colleagues [128], as they observed thattumour cells exhibiting an over fivefold increase in MnSODoverexpression were more sensitive to radiation than controlcells. This was based on an increase in cellular H2O2 concen-tration after detoxification of superoxide radicals after irradia-tion by MnSOD. It has been shown that tumour cells alreadyexhibit elevated levels of ROS and so a reduced detoxifyingcapacity [130]. Indeed we obtained a sixfold to eightfold over-expression rate with our lentiviral vector compared withuntransduced cells. For the AAV vectors, overexpression ratesbelow 5 were observed [118,127], thereby supporting thishypothesis. Similar effects are to be expected by CuZnSOD,yet have not been investigated up to now.

Another possibility is the MnSOD-induced upregulationof the tumour suppressor gene maspin [131], which may bethe reason for a previously observed inversion of the malig-nant phenotype of cells after MnSOD upregulatation [132,133].For TK6 cells overexpressing MnSOD this was not observed,the plating efficiencies between the MnSOD-overexpressing

SNAI2p53

p53

PUMA

p53

HDACSIN3Ax

p53

p53BAX

PUMA

x

x

x

Apoptosis

Irradiation

Cell survivalBCLXL

BCLXL

SNAI2

Figure 5. Model of the anti-apoptotic function of snail

homolog 2 (SNAI2). Due to irradiation transcription of

SNAI2 is induced by p53. SNAI2 in turn activates transcription

of HDAC and switch independent homolog 3A (SIN3A). In

complex with these both proteins SNAI2 binds at regulatory

sequences of the p53 upregulated modulator of apoptosis

(PUMA) gene thus also repressing the p53-induced transcrip-

tion of PUMA. As consequence p53 proteins remains

sequestered by BCLXL, resulting in cell survival. Without

SNAI2 p53-induced PUMA protein interacts with B cell

lymphoma 2-like 1 (BCLXL), the released p53 interacts with

B-cell lymphoma 2-associated X protein (BAX) and thus

induces the mitochondrial apoptotic pathway.Adapted by permission from Macmillan Publishers Ltd: Cell Death and

Differentiation [161], copyright (2006).

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and control cells were not significantly different. This wouldhave been expected due to an inversion of thetumourigenicity by MnSOD.

A further possibility may be the fact that under in vivo con-ditions gene therapeutic overexpression of MnSOD has thepotential to influence inflammation processes. This may beof relevance, as most studies where radioprotection couldbe shown were performed in vivo. Inflammation was shownto have relevance on the side-effects after irradiation, for exam-ple lung fibrosis [134]. Yasui et al. [135] showed that availabilityof SOD at the location of infection can induce H2O2-mediatedapoptosis in activated neutrophils. In another study, Ge et al.[136] demonstrated that the effects of fibrosis after inductionof meningitis in mice could be suppressed by intrathecal appli-cation of SOD. Something similar might also happen in theabove mentioned radioprotection studies in mice: cells of theimmune system (e.g., macrophages and granulocytes) that arenormal recruited to eliminate the tissue damage by the produc-tion of radicals and/or further cytokine-mediated recruitmentof the immune system [137], produce less radicals after SODoverexpression or otherwise inhibit the reactivity of theimmune system. This then may result in reduced collateraldamage during elimination of the focus of inflammation.Results from Rabbani et al. [138] also point in this direction,as they showed that addition of extracellular SOD suppressedmacrophage activity and cytokine production after irradiationand thereby a reduction in fibrosis.

Overall, the observed lack of radioprotection by SOD inmalignant cells may provide an important safety feature forthe use of SOD in future clinical normal tissue radioprotec-tion studies. The first results from a radioprotective Phase Idose-finding study recently showed that oral application ofMnSOD-plasmid/liposomes in patients with NSCLC waswell tolerated [139].

9. Conclusion

In this review, a variety of promising vector systems, radiopro-tective genes and approaches have been described with thegoal of a future clinical implementation of radioprotectionof normal tissue cells. Gene therapeutic radioprotection ofnormal tissue cells certainly has the potential to become ahighly promising novel tool in the battle against cancer.

10. Expert opinion

In recent years also the opposite strategy which is radiosensi-tizing the tumour cells by viral gene therapy showed promis-ing results in clinical trials with two different approaches(Onyx-15, an oncolytic adenovirus, and TNFerade, an adeno-virus with TNF-a under the control of a radio-induciblepromoter) [140,141]. However, a great disadvantage of targetingtumour cells is the requirement of viral transduction of alltumour repopulating cells (tumour stem cells) -- which is infact impossible -- in order to completely eliminate the tumour.

The gene transfer rates will not be high enough to transduceenough cells to obtain a clinically relevant reduction intumour mass. In contrast to this, normal tissue protectionneeds only a low transduction rate since survival of a few nor-mal tissue cells like HSC or intestinal crypt cells is sufficientfor complete regeneration of the damaged tissue. Survival ofonly one crypt stem cell was shown to be enough for develop-ment of a small intestine crypt [142]. The killing of a singletumour stem cell using a sensitizing approach, on the otherhand, would probably have no detectable effect. In our opin-ion, radioprotection is more promising than the radiosensitiz-ing approach, since many of the relevant target areas forradioprotection can be readily accessed by non-invasivemeans, for example oral cavity, pharynx, oesophagus and theintestine (especially the rectum, a primary target for the treat-ment of prostate cancer) as well as HSC mobilized from thebone marrow and isolated from the blood.

Nevertheless, before gene therapeutic radioprotection ofnormal tissue cells can be applied clinically, solid in vivoproof-of-principle has to be shown in small-animal modelsand eventually subsequently in non-human primates. Alsofurther vector optimisation has to be performed. For exampleboth gammaretroviral and lentiviral vectors were shown to beaffected by position dependent variegation compromisinglong-term transgene expression [143]. Although the loss oftransgene expression might be overcome by multiple integra-tions per cell [144], but in order to minimize the risk of inser-tional mutagenesis [72], obtaining only one integration per cellmust be the aim. Thus, incorporation of specific regulatoryelements into the lentiviral vectors represents a prerequisitefor a long-term stable transgene expression in vivo [145]. Forthis reason, special insulator elements [146] or methylationresistant promoters [147,148] were already successfully testedin preclinical approaches. Also a radio-inducible promotercomposed of corresponding elements of the promoter of theEGR1-gene [149] would be beneficial for a distinct expressiononly during the time of radiotherapy, especially for such resis-tance genes whose expression in the long run might supportan oncogenic transformation.

Development of random AAV peptide libraries allowed tar-geting of specific cell types [150,151] by ablating the endogenousundesired AAV-serotype specific tropism and retargeting themto a specific tissue. This technology of cell-specific vector selec-tion is of great importance for the development of targetedgene therapy and might be more beneficial for specific normaltissue protection than the systemic application of small-molecule protectors for which a protective effect on tumourcells cannot be excluded. Pseudotyping of lentiviral vectors byusing a specific envelope protein which interacts with cell-type-specific cell surface proteins might also allow infection ofa restricted cell-type. This strategy has already been successfullyapplied by pseudotyping a lentiviral vector with the envelopeprotein of measles virus, which resulted in preferential and effi-cient infection of CD20+ lymphocytes [152]. Pseudotyped AAVvectors could also be used for targeted infection of cells [153].

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The costs for virus production and for production of small-molecule protectors are probably similar. Various protocolsexist for large-scale good-manufacturing-practice-grade pro-duction of lentiviral vectors [154,155] and of AAV [156]. Sys-temic application of small-molecule protectors or of viralvectors with cell-targeting potential will also require similarexpenses. However, the advantage of the cell-specific ex vivotransduction of mobilized and isolated HSC with subsequenttransplantation will be associated with high costs.

A problem for application of gene therapy for radiopro-tection might be the negative public opinion due to thenegative outcome of previous gene therapy studies. Alreadyin the late 1990s, a serious adverse event was observed in astudy using a first-generation adenoviral vector for thetreatment of ornithine transcarbamylase-deficiency [157].However, intense error analyses revealed that the failurecould be attributed to the use of a first-generation adenovi-ral vector (immunogenic), several errors in the study design(e.g.,: the use of extreme vector dose-escalation withoutproper prior testing), as well as the fact that the patientshould have been excluded from the study due to highurea levels in the blood [158]. Thus, subsequently the for-malities concerning the application for clinical gene therapystudies were defined more exactly. According to concernsabout retroviral vectors, insertional mutagenesis, as men-tioned above, was certainly not causal for induction of leu-kaemia but further genomic instabilities were required.Interestingly, in contrast to SCID-X1, no case of leukaemiaand insertional mutagenesis has been detected in morethan 30 children with ADA-SCID [74]. Thus, it can be sup-posed that HSC of SCID-X1 patients contain in themselvesa higher amount of genomic instabilities than those ofADA-SCID patients. Furthermore, compared to success

rates of 50 -- 85% of allogeneic HSC transplantation whichis the standard care for SCID patients, outcome ofSCID-X1 gene therapy is even better. Concerning AAV-based vectors (not to be confused with AV (adenoviral) vec-tors: in none of the over 80 clinical studies has any seriousadverse effect has been observed. In general, this vector sys-tem is considered relatively safe, primarily due to the lackof any association with any disease, low immunogenicityand the use of ‘gutless’ (having, beside the ITRs, noadditional viral sequences required) vector genomes.

Thus, as a conclusion, we are convinced that radioprotec-tive gene therapy may become a highly promising approachto increase the therapy tolerance due to reduced normal tissuetoxicity. Furthermore, vector optimization for targeted cell-specific transduction and for more stable or regulated trans-gene expression will surely result in improved therapy successand potentially increased quality of life of patients. The searchfor novel radioprotective genes suppressing radiation-induceddamage or supporting DNA-damage repair will increase therange of normal tissue protection. Application of radio-protective gene therapy or of small-molecule protectorsrepresents two competing strategies both with specific prosand cons that, nevertheless, have the potential to be usedin combination.

Acknowledgement

P Maier and MR Veldwijk contributed equally to this paper.

Declaration of interest

The authors state no conflict of interest and have received nopayment in preparation of this manuscript.

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BibliographyPapers of special note have been highlighted as

either of interest (�) or of considerable interest(��) to readers.

1. Ch’ang HJ, Maj JG, Paris F, et al. ATM

regulates target switching to escalating

doses of radiation in the intestines.

Nat Med 2005;11:484-90

2. Dainiak N. Hematologic consequences of

exposure to ionizing radiation.

Exp Hematol 2002;30:513-28

3. Haimovitz-Friedman A, Kolesnick RN,

Fuks Z. Differential inhibition of

radiation-induced apoptosis. Stem Cells

1997;15(Suppl 2):43-7

4. Paris F, Fuks Z, Kang A, et al.

Endothelial apoptosis as the

primary lesion initiating intestinal

radiation damage in mice. Science

2001;293:293-7

5. Scott SD, Greco O. Radiation and

hypoxia inducible gene therapy systems.

Cancer Metastasis Rev 2004;23:269-76

6. Holthusen H. Erfahrungen uber die

Vertraglichkeit von Rontgenstrahlen und

deren Nutzanwendung zur Verhutung

von Schaden. Strahlenther Onkol

1936;57:30-6. This paper describes the fundamentals

of effect/side-effects in radiotherapy.

7. Wenz F, Tiefenbacher U, Willeke F,

Weber KJ. The search for therapeutic

gain in radiation oncology. Onkologie

2001;24(Suppl 5):51-5

8. Bhide SA, Nutting CM. Recent advances

in radiotherapy. BMC Med 2010;8:25

9. Verellen D, Ridder MD, Linthout N,

et al. Innovations in image-guided

radiotherapy. Nat Rev Cancer

2007;7:949-60

10. Page P, Yang LX. Novel

chemoradiosensitizers for cancer therapy.

Anticancer Res 2010;30:3675-82

11. Weiss JF, Landauer MR. History and

development of radiation-protective

agents. Int J Radiat Biol 2009;85:539-73.. A very comprehensive review about

radioprotective agents.

12. Winczura P, Jassem J. Combined

treatment with cytoprotective agents and

radiotherapy. Cancer Treat Rev

2010;36:268-75

13. Savoye C, Swenberg C, Hugot S, et al.

Thiol WR-1065 and disulphide

WR-33278, two metabolites of the drug

ethyol (WR-2721), protect DNA against

fast neutron-induced strand breakage.

Int J Radiat Biol 1997;71:193-202

14. Sasse AD, Clark LG, Sasse EC,

Clark OA. Amifostine reduces side effects

and improves complete response rate

during radiotherapy: results of a

meta-analysis. Int J Radiat Oncol

Biol Phys 2006;64:784-91

15. Mell LK, Malik R, Komaki R, et al.

Effect of amifostine on response rates in

locally advanced non-small-cell lung

cancer patients treated on randomized

controlled trials: a meta-analysis. Int J

Radiat Oncol Biol Phys 2007;68:111-18

16. Finch PW, Rubin JS, Miki T, et al.

Human KGF is FGF-related with

properties of a paracrine effector of

epithelial cell growth. Science

1989;245:752-5

17. Finch PW, Rubin JS. Keratinocyte

growth factor expression and activity in

cancer: implications for use in patients

with solid tumors. J Natl Cancer Inst

2006;98:812-24

18. Farrell CL, Bready JV, Rex KL, et al.

Keratinocyte growth factor protects

mice from chemotherapy and

radiation-induced gastrointestinal injury

and mortality. Cancer Res 1998;58:933-9

19. Bohlius J, Schmidlin K, Brillant C,

et al. Recombinant human

erythropoiesis-stimulating agents and

mortality in patients with cancer:

a meta-analysis of randomised trials.

Lancet 2009;373:1532-42

20. Lambin P, Ramaekers BL,

van Mastrigt GA, et al. Erythropoietin as

an adjuvant treatment with (chemo)

radiation therapy for head and neck

cancer. Cochrane Database Syst Rev

2009:CD006158

21. Ryu JK, Swann S, LeVeque F, et al.

The impact of concurrent granulocyte

macrophage-colony stimulating factor on

radiation-induced mucositis in head and

neck cancer patients: a double-blind

placebo-controlled prospective Phase III

study by Radiation Therapy Oncology

Group 9901. Int J Radiat Oncol

Biol Phys 2007;67:643-50

22. Su YB, Vickers AJ, Zelefsky MJ, et al.

Double-blind, placebo-controlled,

randomized trial of granulocyte-colony

stimulating factor during postoperative

radiotherapy for squamous head and

neck cancer. Cancer J 2006;12:182-8

23. Jiang J, McDonald PR, Dixon TM, et al.

Synthetic protection short interfering

RNA screen reveals glyburide as a novel

radioprotector. Radiat Res

2009;172:414-22

24. Thotala DK, Geng L, Dickey AK, et al.

A new class of molecular targeted

radioprotectors: GSK-3beta inhibitors.

Int J Radiat Oncol Biol Phys

2010;76:557-65

25. Epperly MW, Goff JP, Li S, et al.

Intraesophageal administration of

GS-nitroxide (JP4-039) protects against

ionizing irradiation-induced esophagitis.

In Vivo 2010;24:811-19

26. Jiang J, Stoyanovsky DA, Belikova NA,

et al. A mitochondria-targeted

triphenylphosphonium-conjugated

nitroxide functions as a radioprotector/

mitigator. Radiat Res 2009;172:706-17

27. Kim K, Pollard JM, Norris AJ, et al.

High-throughput screening identifies two

classes of antibiotics as radioprotectors:

tetracyclines and fluoroquinolones.

Clin Cancer Res 2009;15:7238-45

28. Camphausen K, Tofilon PJ. Combining

radiation and molecular targeting in

cancer therapy. Cancer Biol Ther

2004;3:247-50

29. Gridley DS, Slater JM. Combining gene

therapy and radiation against cancer.

Curr Gene Ther 2004;4:231-48

30. Flotte TR. Gene therapy: the first two

decades and the current state-of-the-art.

J Cell Physiol 2007;213:301-5. An interesting read on the history

and current state-of-the-art of

gene therapy.

31. Wells DJ. Electroporation and

ultrasound enhanced non-viral gene

delivery in vitro and in vivo.

Cell Biol Toxicol 2010;26:21-8

32. Wolff JA, Budker V. The mechanism of

naked DNA uptake and expression.

Adv Genet 2005;54:3-20

33. Kootstra NA, Verma IM. Gene therapy

with viral vectors. Annu Rev

Pharmacol Toxicol 2003;43:413-39

34. Gene Therapy Clinical Trials Worldwide.

John Wiley and Sons, Hoboken, NJ;

2007. Available from: http://www.wiley.

com/legacy/wileychi/genmed/clinical/

[Last accessed 20 April 2011]. A good database for information on all

clinical gene therapy studies.

Radioprotective gene therapy

1146 Expert Opin. Biol. Ther. (2011) 11(9)

Exp

ert O

pin.

Bio

l. T

her.

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

RM

IT U

nive

rsity

on

02/2

2/13

For

pers

onal

use

onl

y.

35. Blacklow NR, Hoggan MD, Sereno MS,

et al. A seroepidemiologic study of

adenovirus-associated virus infection in

infants and children. Am J Epidemiol

1971;94:359-66

36. Handa H, Carter BJ. Adeno-associated

virus DNA replication complexes in

herpes simplex virus or

adenovirus-infected cells. J Biol Chem

1979;254:6603-10

37. Hoggan MD, Blacklow NR, Rowe WP.

Studies of small DNA viruses found in

various adenovirus preparations: physical,

biological, and immunological

characteristics. Proc Natl Acad Sci USA

1966;55:1467-74

38. Srivastava A, Lusby EW, Berns KI.

Nucleotide sequence and organization of

the adeno-associated virus 2 genome.

J Virol 1983;45:555-64

39. Hauswirth WW, Berns KI. Origin and

termination of adeno-associated virus

DNA replication. Virology

1977;78:488-99

40. Berns KI, Kotin RM, Labow MA.

Regulation of adeno-associated virus

DNA replication. Biochim Biophys Acta

1988;951:425-9

41. Rose JA, Koczot F. Adenovirus-associated

virus multiplication. VI. Base compostion

of the deoxyribonucleic acid strand

species and strand-specific in vivo

transcription. J Virol 1971;8:771-7

42. Wistuba A, Weger S, Kern A,

Kleinschmidt JA. Intermediates of

adeno-associated virus type 2 assembly:

identification of soluble complexes

containing Rep and Cap proteins. J Virol

1995;69:5311-19

43. Sonntag F, Schmidt K, Kleinschmidt JA.

A viral assembly factor promotes

AAV2 capsid formation in the nucleolus.

Proc Natl Acad Sci USA

2010;107:10220-5

44. Gao GP, Alvira MR, Wang L, et al.

Novel adeno-associated viruses from

rhesus monkeys as vectors for human

gene therapy. Proc Natl Acad Sci USA

2002;99:11854-9

45. Halbert CL, Rutledge EA, Allen JM,

et al. Repeat transduction in the mouse

lung by using adeno-associated virus

vectors with different serotypes. J Virol

2000;74:1524-32

46. Bantel-Schaal U. Adeno-associated

parvoviruses inhibit growth of cells

derived from malignant human tumors.

IntJCancer 1990;45:190-4

47. Hermonat PL. Adeno-associated virus

inhibits human papillomavirus type 16:

a viral interaction implicated in cervical

cancer. Cancer Res 1994;54:2278-81

48. Walz CM, Anisi TR, Schlehofer JR,

et al. Detection of infectious

adeno-associated virus particles in

human cervical biopsies. Virology

1998;247:97-105

49. Batshaw ML, Wilson JM, Raper S, et al.

Recombinant adenovirus gene transfer in

adults with partial ornithine

transcarbamylase deficiency (OTCD).

Hum Gene Ther 1999;10:2419-37

50. Dyall J, Szabo P, Berns KI.

Adeno-associated virus (AAV)

site-specific integration: formation of

AAV-AAVS1 junctions in an in vitro

system. Proc Natl Acad Sci USA

1999;96:12849-54

51. Philpott NJ, Gomos J, Berns KI,

Falck-Pedersen E. A p5 integration

efficiency element mediates

Rep-dependent integration into

AAVS1 at chromosome 19. Proc Natl

Acad Sci USA 2002;99:12381-5

52. Hughes SM, Moussavi-Harami F,

Sauter SL, Davidson BL. Viral-mediated

gene transfer to mouse primary neural

progenitor cells. MolTher 2002;5:16-24

53. Flotte TR, Zeitlin PL, Reynolds TC,

et al. Phase I trial of intranasal and

endobronchial administration of a

recombinant adeno-associated virus

serotype 2 (rAAV2)-CFTR vector in

adult cystic fibrosis patients: a two-part

clinical study. Hum Gene Ther

2003;14:1079-88

54. Moss RB, Milla C, Colombo J, et al.

Repeated aerosolized AAV-CFTR for

treatment of cystic fibrosis: a randomized

placebo-controlled Phase 2B trial.

Hum Gene Ther 2007;18:726-32

55. Jiang H, Pierce GF, Ozelo MC, et al.

Evidence of multiyear factor IX

expression by AAV-mediated gene

transfer to skeletal muscle in an

individual with severe hemophilia B.

Mol Ther 2006;14:452-5

56. Manno CS, Pierce GF, Arruda VR, et al.

Successful transduction of liver in

hemophilia by AAV-Factor IX and

limitations imposed by the host

immune response. Nat Med

2006;12:342-7

57. Niemeyer GP, Herzog RW, Mount J,

et al. Long-term correction of

inhibitor-prone hemophilia B

dogs treated with liver-directed

AAV2-mediated factor IX gene therapy.

Blood 2009;113:797-806

58. Veldwijk MR, Sellner L, Stiefelhagen M,

et al. Pseudotyped recombinant

adeno-associated viral vectors mediate

efficient gene transfer into primary

human CD34+ peripheral blood

progenitor cells. Cytotherapy

2010;12:107-12. This paper describes the use of

pseudotyped AAV vectors to increase

specificity and selectivity of vectors

towards a specific tissue.

59. Bankiewicz KS, Forsayeth J, Eberling JL,

et al. Long-term clinical improvement in

MPTP-lesioned primates after gene

therapy with AAV-hAADC. Mol Ther

2006;14:564-70

60. Lewitt PA, Rezai AR, Leehey MA, et al.

AAV2-GAD gene therapy for advanced

Parkinson’s disease: a double-blind,

sham-surgery controlled, randomised

trial. Lancet Neurol 2011;10:309-19.. Successful clinical study using

AAV-based vectors for the treatment of

Parkinson’s disease.

61. Muramatsu S, Fujimoto K, Kato S, et al.

A Phase I study of aromatic L-amino

acid decarboxylase gene therapy for

Parkinson’s disease. Mol Ther

2010;18:1731-5

62. Acland GM, Aguirre GD, Ray J, et al.

Gene therapy restores vision in a canine

model of childhood blindness. Nat Genet

2001;28:92-5. Proof of principle for correction of

LCA using AAV-based vectors in a dog

model. The basis for later

clinical studies.

63. Le Meur G, Stieger K, Smith AJ, et al.

Restoration of vision in RPE65-deficient

Briard dogs using an AAV serotype 4

vector that specifically targets the retinal

pigmented epithelium. Gene Ther

2007;14:292-303

64. Bainbridge JW, Smith AJ, Barker SS,

et al. Effect of gene therapy on visual

function in Leber’s congenital amaurosis.

N Engl J Med 2008;358:2231-9

65. Maguire AM, Simonelli F, Pierce EA,

et al. Safety and efficacy of gene transfer

for Leber’s congenital amaurosis. N Engl

J Med 2008;358:2240-8

Maier, Veldwijk & Wenz

Expert Opin. Biol. Ther. (2011) 11(9) 1147

Exp

ert O

pin.

Bio

l. T

her.

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

RM

IT U

nive

rsity

on

02/2

2/13

For

pers

onal

use

onl

y.

66. Cideciyan AV, Hauswirth WW,

Aleman TS, et al. Human RPE65 gene

therapy for Leber congenital amaurosis:

persistence of early visual improvements

and safety at 1 year. Hum Gene Ther

2009;20:999-1004

67. Simonelli F, Maguire AM, Testa F, et al.

Gene therapy for Leber’s congenital

amaurosis is safe and effective through

1.5 years after vector administration.

Mol Ther 2010;18:643-50

68. Miller AD, Rosman GJ. Improved

retroviral vectors for gene transfer and

expression. Biotechniques

1989;7:980-2, 984-986, 989-990

69. Schambach A, Baum C. Clinical

application of lentiviral vectors - concepts

and practice. Curr Gene Ther

2008;8:474-82

70. Fouchier RA, Malim MH. Nuclear

import of human immunodeficiency

virus type-1 preintegration complexes.

Adv Virus Res 1999;52:275-99

71. Maier P, von Kalle C, Laufs S.

Retroviral vectors for gene therapy.

Future Microbiol 2010;5:1507-23

72. Aiuti A, Roncarolo MG. Ten years of

gene therapy for primary immune

deficiencies. Hematology Am Soc

Hematol Educ Program 2009:682-9

73. Cartier N, Hacein-Bey-Abina S,

Bartholomae CC, et al. Hematopoietic

stem cell gene therapy with a lentiviral

vector in X-linked adrenoleukodystrophy.

Science 2009;326:818-23

74. Sokolic R, Kesserwan C, Candotti F.

Recent advances in gene therapy for

severe congenital immunodeficiency

diseases. Curr Opin Hematol

2008;15:375-80

75. Kohn DB, Candotti F. Gene therapy

fulfilling its promise. N Engl J Med

2009;360:518-21

76. Qasim W, Gaspar HB, Thrasher AJ.

Progress and prospects: gene therapy for

inherited immunodeficiencies. Gene Ther

2009;16:1285-91

77. Hacein-Bey-Abina S, Garrigue A,

Wang GP, et al. Insertional oncogenesis

in 4 patients after retrovirus-mediated

gene therapy of SCID-X1. J Clin Invest

2008;118:3132-42

78. Howe SJ, Mansour MR,

Schwarzwaelder K, et al. Insertional

mutagenesis combined with acquired

somatic mutations causes leukemogenesis

following gene therapy of

SCID-X1 patients. J Clin Invest

2008;118:3143-50

79. Fruehauf S, Klaus J, Huesing J, et al.

Efficient mobilization of peripheral blood

stem cells following CAD chemotherapy

and a single dose of pegylated G-CSF in

patients with multiple myeloma.

Bone Marrow Transplant

2007;39:743-50

80. Fruehauf S, Ehninger G, Hubel K, et al.

Mobilization of peripheral blood stem

cells for autologous transplant in non-

Hodgkin’s lymphoma and multiple

myeloma patients by plerixafor and

G-CSF and detection of tumor cell

mobilization by PCR in multiple

myeloma patients.

Bone Marrow Transplant

2010;45:269-75

81. Fruehauf S, Haas R, Zeller WJ,

Hunstein W. CD34 selection for purging

in multiple myeloma and analysis of

CD34+ B cell precursors. Stem Cells

1994;12:95-102

82. Civin CI, Strauss LC, Brovall C, et al.

Antigenic analysis of hematopoiesis. III.

A hematopoietic progenitor cell surface

antigen defined by a monoclonal

antibody raised against KG-1a cells.

J Immunol 1984;133:157-65

83. Sorrentino BP. Gene therapy to protect

haematopoietic cells from cytotoxic

cancer drugs. Nat Rev Cancer

2002;2:431-41

84. Gottesman MM, Ling V. The molecular

basis of multidrug resistance in

cancer: the early years of P-glycoprotein

research. FEBS Lett

2006;580:998-1009

85. Chaudhary PM, Roninson IB. Expression

and activity of P-glycoprotein, a

multidrug efflux pump, in human

hematopoietic stem cells. Cell

1991;66:85-94

86. van Tellingen O. The importance of

drug-transporting P-glycoproteins in

toxicology. Toxicol Lett

2001;120:31-41

87. Fruehauf S, Breems DA,

Knaan-Shanzer S, et al. Frequency

analysis of multidrug resistance-1 gene

transfer into human primitive

hematopoietic progenitor cells

using the cobblestone area-forming cell

assay and detection of vector-mediated

P-glycoprotein expression by

rhodamine-123. Human Gene Ther

1996;7:1219-31

88. Becker PS, Yu YP, Ceredona AM, et al.

Transfer of the multidrug resistance gene,

MDR-1, to hematopoietic progenitors

from a patient with transformed large

cell lymphoma, and demonstration of

in vivo chemotherapy protection with

dose escalating chemotherapy. Blood

2004;104:5278

89. Takahashi S, Aiba K, Ito Y, et al. Pilot

study of MDR1 gene transfer into

hematopoietic stem cells and

chemoprotection in metastatic breast

cancer patients. Cancer Sci

2007;98:1609-16

90. Ruefli AA, Tainton KM, Darcy PK,

et al. P-glycoprotein inhibits

caspase-8 activation but not formation of

the death inducing signal complex (disc)

following Fas ligation. Cell Death Differ

2002;9:1266-72. A report about the antiapoptotic

function of MDR1.

91. Tainton KM, Smyth MJ, Jackson JT,

et al. Mutational analysis of

P-glycoprotein: suppression of caspase

activation in the absence of

ATP-dependent drug efflux.

Cell Death Differ 2004;11:1028-37

92. Maier P, Fleckenstein K, Li L, et al.

Overexpression of MDR1 using a

retroviral vector differentially regulates

genes involved in detoxification and

apoptosis and confers radioprotection.

Radiat Res 2006;166:463-73. A report about radioprotection by

lentiviral overexpression of MDR1.

93. Maier P, Herskind C, Fleckenstein K,

et al. MDR1 gene transfer using a

lentiviral SIN vector confers

radioprotection to human CD34+

hematopoietic progenitor cells.

Radiat Res 2008;169:301-10

94. Schiedlmeier B, Kuhlcke K, Eckert HG,

et al. Quantitative assessment of

retroviral transfer of the human

multidrug resistance 1 gene to human

mobilized peripheral blood progenitor

cells engrafted in nonobese diabetic/

severe combined immunodeficient mice.

Blood 2000;95:1237-48

95. De Craene B, van Roy F, Berx G.

Unraveling signalling cascades for the

Snail family of transcription factors.

Cell Signal 2005;17:535-47

96. Inoue A, Seidel MG, Wu W, et al.

Slug, a highly conserved zinc finger

transcriptional repressor, protects

hematopoietic progenitor cells from

Radioprotective gene therapy

1148 Expert Opin. Biol. Ther. (2011) 11(9)

Exp

ert O

pin.

Bio

l. T

her.

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

RM

IT U

nive

rsity

on

02/2

2/13

For

pers

onal

use

onl

y.

radiation-induced apoptosis in vivo.

Cancer Cell 2002;2:279-88. A report about the antiapoptotic

function of Slug/SNAI2 in

haematopoietic stem cells.

97. Qiu W, Carson-Walter EB, Liu H, et al.

PUMA regulates intestinal progenitor cell

radiosensitivity and gastrointestinal

syndrome. Cell Stem Cell 2008;2:576-83

98. Wu WS, Heinrichs S, Xu D, et al. Slug

antagonizes p53-mediated apoptosis of

hematopoietic progenitors by repressing

puma. Cell 2005;123:641-53

99. Chipuk JE, Bouchier-Hayes L,

Kuwana T, et al. PUMA couples the

nuclear and cytoplasmic proapoptotic

function of p53. Science

2005;309:1732-5

100. Mustata G, Li M, Zevola N, et al.

Development of small-molecule

PUMA inhibitors for mitigating

radiation-induced cell death. Curr Top

Med Chem 2011;11:281-90

101. Maier P, Herskind C, Barzan D, et al.

SNAI2 as a novel radioprotector of

normal tissue by gene transfer using a

lentiviral bicistronic SIN vector.

Radiat Res 2010;173:612-19

102. Schafer J, Bachtler J, Engling A, et al.

Suppression of apoptosis and clonogenic

survival in irradiated human

lymphoblasts with different TP53 status.

Radiat Res 2002;158:699-706

103. Yu Y, Little JB. p53 is involved in but

not required for ionizing

radiation-induced caspase-3 activation

and apoptosis in human lymphoblast cell

lines. Cancer Res 1998;58:4277-81

104. Keele BB Jr, McCord JM, Fridovich I.

Superoxide dismutase from Escherichia

coli B. A new manganese-containing

enzyme. J Biol Chem 1970;245:6176-81

105. Mann T, Keilin D. Haemocuprein and

hepatocuprein, copper-protein compound

of blood and liver in mammals. Proc R

Soc London Ser B Biol Sci

1939;126:303-15

106. McCord JM, Fridovich I. Superoxide

dismutase. An enzymic function for

erythrocuprein (hemocuprein).

J Biol Chem 1969;244:6049-55

107. Marklund SL, Holme E, Hellner L.

Superoxide dismutase in extracellular

fluids. Clin Chim Acta 1982;126:41-51

108. Akashi M, Hachiya M, Paquette RL,

et al. Irradiation increases manganese

superoxide dismutase mRNA levels in

human fibroblasts. Possible mechanisms

for its accumulation. J Biol Chem

1995;270:15864-9

109. Hardmeier R, Hoeger H,

Fang-Kircher S, et al. Transcription and

activity of antioxidant enzymes after

ionizing irradiation in radiation-resistant

and radiation-sensitive mice. Proc Natl

Acad Sci USA 1997;94:7572-6

110. Riley PA. Free radicals in biology:

oxidative stress and the effects of ionizing

radiation. Int J Radiat Biol

1994;65:27-33

111. Vozenin-Brotons MC, Sivan V, Gault N,

et al. Antifibrotic action of Cu/Zn SOD

is mediated by TGF-beta1 repression and

phenotypic reversion of myofibroblasts.

Free Radic Biol Med 2001;30:30-42

112. Epperly MW, Sikora CA, DeFilippi SJ,

et al. Manganese superoxide dismutase

(SOD2) inhibits radiation-induced

apoptosis by stabilization of the

mitochondrial membrane. Radiat Res

2002;157:568-77

113. Noshita N, Sugawara T, Fujimura M,

et al. Manganese superoxide dismutase

affects cytochrome c release and

caspase-9 activation after transient focal

cerebral ischemia in mice. J Cereb Blood

Flow Metab 2001;21:557-67

114. Drane P, Bravard A, Bouvard V, May E.

Reciprocal down-regulation of p53 and

SOD2 gene expression-implication in

p53 mediated apoptosis. Oncogene

2001;20:430-9

115. Epperly MW, Bray JA, Krager S, et al.

Intratracheal injection of adenovirus

containing the human MnSOD

transgene protects athymic nude mice

from irradiation-induced organizing

alveolitis. Int J Radiat Oncol Biol Phys

1999;43:169-81

116. Sun J, Chen Y, Li M, Ge Z. Role of

antioxidant enzymes on ionizing

radiation resistance. Free Radic Biol Med

1998;24:586-93

117. Delanian S, Martin M, Bravard A, et al.

Cu/Zn superoxide dismutase modulates

phenotypic changes in cultured

fibroblasts from human skin with chronic

radiotherapy damage. Radiother Oncol

2001;58:325-31

118. Veldwijk MR, Herskind C, Sellner L,

et al. Normal-tissue radioprotection by

overexpression of the copper-zinc and

manganese superoxide dismutase genes.

Strahlenther Onkol 2009;185:517-23

119. Epperly MW, Defilippi S, Sikora C,

et al. Intratracheal injection of

manganese superoxide dismutase

(MnSOD) plasmid/liposomes protects

normal lung but not orthotopic tumors

from irradiation. Gene Ther

2000;7:1011-18

120. Stickle RL, Epperly MW, Klein E, et al.

Prevention of irradiation-induced

esophagitis by plasmid/liposome delivery

of the human manganese superoxide

dismutase transgene.

Radiat Oncol Investig 1999;7:204-17

121. Epperly MW, Tyurina YY, Nie S, et al.

MnSOD-plasmid liposome gene therapy

decreases ionizing irradiation-induced

lipid peroxidation of the esophagus.

In Vivo 2005;19:997-1004

122. Epperly MW, Kagan VE, Sikora CA,

et al. Manganese superoxide

dismutase-plasmid/liposome

(MnSOD-PL) administration protects

mice from esophagitis associated with

fractionated radiation. Int J Cancer

2001;96:221-31

123. Epperly MW, Sikora C, Defilippi S,

et al. Plasmid/liposome transfer of the

human manganese superoxide dismutase

transgene prevents ionizing

irradiation-induced apoptosis in human

esophagus organ explant culture.

Int J Cancer 2000;90:128-37

124. Kanai AJ, Zeidel ML, Lavelle JP, et al.

Manganese superoxide dismutase

gene therapy protects against

irradiation-induced cystitis. Am J Physiol

Renal Physiol 2002;283:F1304-12

125. Guo H, Seixas-Silva JA Jr, Epperly MW,

et al. Prevention of radiation-induced

oral cavity mucositis by plasmid/liposome

delivery of the human manganese

superoxide dismutase (SOD2) transgene.

Radiat Res 2003;159:361-70

126. Epperly MW, Epperly LD, Niu Y, et al.

Overexpression of the MnSOD transgene

product protects cryopreserved bone

marrow hematopoietic progenitor cells

from ionizing radiation. Radiat Res

2007;168:560-6

127. Veldwijk MR, Herskind C, Laufs S,

et al. Recombinant adeno-associated virus

2-mediated transfer of the human

superoxide-dismutase gene does not

confer radioresistance on HeLa cervical

carcinoma cells. Radiother Oncol

2004;72:341-50

128. Zhong W, Oberley LW, Oberley TD,

et al. Inhibition of cell growth and

Maier, Veldwijk & Wenz

Expert Opin. Biol. Ther. (2011) 11(9) 1149

Exp

ert O

pin.

Bio

l. T

her.

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

RM

IT U

nive

rsity

on

02/2

2/13

For

pers

onal

use

onl

y.

sensitization to oxidative damage by

overexpression of manganese superoxide

dismutase in rat glioma cells.

Cell Growth Differ 1996;7:1175-86

129. Borrelli A, Schiattarella A, Mancini R,

et al. A recombinant MnSOD is

radioprotective for normal cells and

radiosensitizing for tumor cells.

Free Radic Biol Med 2009;46:110-16

130. Aykin-Burns N, Ahmad IM, Zhu Y,

et al. Increased levels of superoxide and

H2O2 mediate the differential

susceptibility of cancer cells versus

normal cells to glucose deprivation.

Biochem J 2009;418:29-37

131. Duan H, Zhang HJ, Yang JQ, et al.

MnSOD up-regulates maspin tumor

suppressor gene expression in human

breast and prostate cancer cells.

Antioxid Redox Signal 2003;5:677-88

132. Amstad PA, Liu H, Ichimiya M, et al.

Manganese superoxide dismutase

expression inhibits soft agar growth in

JB6 clone41 mouse epidermal cells.

Carcinogenesis 1997;18:479-84

133. Urano M, Kuroda M, Reynolds R, et al.

Expression of manganese superoxide

dismutase reduces tumor control

radiation dose: gene-radiotherapy.

Cancer Res 1995;55:2490-3

134. Fleckenstein K, Gauter-Fleckenstein B,

Jackson IL, et al. Using biological

markers to predict risk of radiation

injury. Semin Radiat Oncol

2007;17:89-98

135. Yasui K, Kobayashi N, Yamazaki T,

et al. Superoxide dismutase (SOD) as a

potential inhibitory mediator of

inflammation via neutrophil apoptosis.

Free Radic Res 2005;39:755-62

136. Ge NN, Brodie SA, Tinling SP,

Brodie HA. The effects of superoxide

dismutase in gerbils with bacterial

meningitis. Otolaryngol Head Neck Surg

2004;131:563-72

137. Rubin P, Johnston CJ, Williams JP,

et al. A perpetual cascade of cytokines

postirradiation leads to pulmonary

fibrosis. Int J Radiat Oncol Biol Phys

1995;33:99-109

138. Rabbani ZN, Anscher MS, Folz RJ, et al.

Overexpression of extracellular superoxide

dismutase reduces acute radiation

induced lung toxicity. BMC Cancer

2005;5:59

139. Tarhini AA, Belani CP, Luketich JD,

et al. A Phase I study of concurrent

chemotherapy (paclitaxel and

carboplatin) and thoracic radiotherapy

with swallowed manganese superoxide

dismutase (MnSOD) plasmid liposome

(PL) protection in patients with locally

advanced stage III non-small cell lung

cancer. Human Gene Ther

2011;22:336-42

140. Kumar S, Gao L, Yeagy B, Reid T. Virus

combinations and chemotherapy for the

treatment of human cancers. Curr Opin

Mol Ther 2008;10:371-9

141. Weichselbaum RR, Kufe D. Translation

of the radio- and chemo-inducible

TNFerade vector to the treatment of

human cancers. Cancer Gene Ther

2009;16:609-19

142. Potten CS. A comprehensive study of the

radiobiological response of the murine

(BDF1) small intestine. Int J Radiat Biol

1990;58:925-73

143. Ellis J. Silencing and variegation of

gammaretrovirus and lentivirus vectors.

Hum Gene Ther 2005;16:1241-6

144. Woods NB, Muessig A, Schmidt M,

et al. Lentiviral vector transduction of

NOD/SCID repopulating cells

results in multiple vector integrations

per transduced cell: risk of

insertional mutagenesis. Blood

2003;101:1284-9

145. Hawley RG. Progress toward vector

design for hematopoietic stem

cell gene therapy. Curr Gene Ther

2001;1:1-17

146. Ramezani A, Hawley TS, Hawley RG.

Performance- and safety-enhanced

lentiviral vectors containing the human

interferon-beta scaffold attachment region

and the chicken beta-globin insulator.

Blood 2003;101:4717-24

147. Almarza E, Zhang F, Santilli G, et al.

Correction of SCID-X1 using an

enhancerless vav promoter.

Hum Gene Ther 2011;22:263-70

148. Zhang F, Thornhill SI, Howe SJ, et al.

Lentiviral vectors containing an

enhancer-less ubiquitously acting

chromatin opening element (UCOE)

provide highly reproducible and stable

transgene expression in hematopoietic

cells. Blood 2007;110:1448-57. A report about a special regulatory

element for stable transgene expression

by lentiviral vectors.

149. Marignol L, Coffey M, Hollywood D,

Lawler M. Radiation to control transgene

expression in tumors. Cancer Biol Ther

2007;6:1005-12

150. Muller OJ, Kaul F, Weitzman MD,

et al. Random peptide libraries displayed

on adeno-associated virus to select for

targeted gene therapy vectors.

Nat Biotechnol 2003;21:1040-6

151. Sellner L, Stiefelhagen M,

Kleinschmidt JA, et al. Generation of

efficient human blood

progenitor-targeted recombinant

adeno-associated viral vectors (AAV) by

applying an AAV random peptide library

on primary human hematopoietic

progenitor cells. Exp Hematol

2008;36:957-64

152. Funke S, Maisner A, Muhlebach MD,

et al. Targeted cell entry of lentiviral

vectors. Mol Ther 2008;16:1427-36

153. Veldwijk MR, Sellner L, Stiefelhagen M,

et al. Pseudotyped recombinant

adeno-associated viral vectors mediate

efficient gene transfer into primary

human CD34+ peripheral blood

progenitor cells. Cytotherapy

2010;12:107-112

154. Segura MM, Garnier A, Durocher Y,

et al. New protocol for lentiviral

vector mass production.

Methods Mol Biol 2010;614:39-52

155. Schweizer M, Merten OW. Large-scale

production means for the manufacturing

of lentiviral vectors. Curr Gene Ther

2010;10:474-86

156. Cecchini S, Negrete A, Kotin RM.

Toward exascale production of

recombinant adeno-associated virus for

gene transfer applications. Gene Ther

2008;15:823-30

157. Raper SE, Chirmule N, Lee FS, et al.

Fatal systemic inflammatory response

syndrome in a ornithine transcarbamylase

deficient patient following adenoviral

gene transfer. Mol Genet Metab

2003;80:148-58

158. Somia N, Verma IM. Gene therapy:

trials and tribulations. Nat Rev Genet

2000;1:91-9.. An interesting read on the first gene

therapy-related death and

the consequences.

159. Kotin RM. Prospects for the use of

adeno-associated virus as a vector for

human gene therapy. Hum Gene Ther

1994;5:793-801

160. Zolotukhin S, Potter M,

Hauswirth WW, et al. A “humanized”

Radioprotective gene therapy

1150 Expert Opin. Biol. Ther. (2011) 11(9)

Exp

ert O

pin.

Bio

l. T

her.

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

RM

IT U

nive

rsity

on

02/2

2/13

For

pers

onal

use

onl

y.

green fluorescent protein cDNA adapted

for high-level expression in mammalian

cells. J Virol 1996;70:4646-54

161. Haupt S, Alsheich-Bartok O, Haupt Y.

Clues from worms: a Slug at Puma

promotes the survival of blood

progenitors. Cell Death Differ

2006;13:913-15

AffiliationPatrick Maier†, Marlon R Veldwijk &

Frederik Wenz†Author for correspondence

Heidelberg University,

University Medical Centre Mannheim,

Department of Radiation Oncology,

Theodor-Kutzer-Ufer 1-3,

68167 Mannheim, Germany

Tel: +0049 6213833773;

E-mail: patrick.maier@medma.uni-heidelberg.de

Maier, Veldwijk & Wenz

Expert Opin. Biol. Ther. (2011) 11(9) 1151

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Dow

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from

info

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care

.com

by

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on

02/2

2/13

For

pers

onal

use

onl

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