Integration of retroviral vectors

Integration of retroviral vectorsRichard Gabriel, Manfred Schmidt and Christof von Kalle

Available online at www.sciencedirect.com

Retroviral vectors are unique in their ability to integrate their

genome into the host genome of transduced cells. Several

members of the retrovirus family show distinct pattern for

preferential integration into the host genome. Despite many

years of investigation, precise mechanisms of target site

selection and the fundamental interplay of viral integrase and

host cell proteins are still unknown. Improved methods to detect

retroviral integrations genome-wide as well as recent advances

on the retroviral integrase structure and integrase interacting

proteins may lead to further uncover the process of retroviral

target site selection. A better knowledge of these mechanisms

and interactions will allow further improving safety of retroviral

vectors for gene therapy by providing an opportunity to retarget

retroviral integration into non-harmful genomic positions.

Address

Department of Translational Oncology, National Center for Tumor

Diseases (NCT) and German Cancer Research Center (DKFZ), Im

Neuenheimer Feld 460, 69120 Heidelberg, Germany

Corresponding author: von Kalle, Christof

([email protected])

Current Opinion in Immunology 2012, 24:592–597

This review comes from a themed issue on Immunogenetics and

transplantation

Edited by Alain Fischer and Matthew Porteus

For a complete overview see the Issue and the Editorial

Available online 14th September 2012

0952-7915/$ – see front matter, # 2012 Elsevier Ltd. All rights

reserved.

http://dx.doi.org/10.1016/j.coi.2012.08.006

IntroductionThe family of retroviruses consists of two subfamilies:

Orthoretrovirinae with the genera of simple alpha-retro-

viruses, beta-retroviruses, gammaretroviruses and com-

plex delta-viruses, epsilon-viruses and lentiviruses as well

as Spumaretrovirinae with the genus of the foamy viruses.

Integration of the viral genome is a hallmark for the

retroviral life cycle. Because of the integration into the

host genome the virus becomes an inheritable part of the

host cell. This feature renders retrovirus-derived vectors

attractive tools as gene transfer vehicles, as they stably

introduce functional genes or new genetic information

into the host cell which are then expressed from the

integrated provirus.

The various genera exhibit different features regarding

cell tropism and integration preference. One major


difference is the ability to transduce resting or cycling

cells. Gammaretroviral vectors are dependent on

degeneration of the nuclear membrane during cell

division to allow the virus to enter the nucleus. In con-

trast, lentiviral vectors actively enter the nucleus via the

nuclear pore enabling them to transduce non-dividing

cells efficiently. Despite many years of investigation, the

exact interplay between viral integrase (IN) and host

proteins and how cellular cofactors modulate target

site selection of the different genera remain largely

unknown.

Understanding the mechanisms underlying retroviral

integration as well as identifying positions in the host

genome where retroviral vectors integrate are crucial to

further improve retroviral vectors for safe gene thera-

peutic applications. This review focuses on recent

advances in understanding the integration mechanism

of retroviral vectors, new strategies to indentify provirus

locations in the host genome and the consequences of

integrated provirus on the host cell.

Detection of retroviral integration lociIntegration of retroviruses and derived vectors is

mediated by the viral IN encoded by the pol gene.

Retroviral integration has been extensively studied in

the past (reviewed in [1]). IN assembles with viral

DNA and cellular proteins to form the preintegration

complex (PIC). IN possesses two catalytic activities: 30

end processing, cleaving a dinucleotide from the 30 end of

each long terminal repeat (LTR) leaving an invariant CA

dinucleotide and DNA strand transfer which uses the

recessed 30 termini to open the target DNA to join the

linear viral genome to the host genome. Integration is

completed by host DNA repair enzymes, resulting in

species-specific 4–6 bp direct repeats flanking the

provirus (reviewed in [2]).

Several PCR-based methods have been developed to

efficiently amplify and identify retroviral integration

sites (IS) from the host genome, and most prominent

among those are LAM-PCR [3] and LM-PCR (in-

cluding several modifications) [4,5]. All conventional

IS analysis methods are dependent on restriction

enzymes to digest the genomic DNA before ligation

of a known linker sequence that allows subsequent

amplification and sequencing of the vector genome

junctions. Mapping of the genomic amplicon sequences

enables us to precisely locate retroviral IS in the host

genome. However, applying optimal restriction enzyme

combinations is crucial to circumvent restriction and

amplification biases [6].

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Integration of retroviral vectors Gabriel, Schmidt and von Kalle 593

Recently developed restriction enzyme independent

methods uncover retroviral IS either by the ligation of

single-strand oligonucleotides to amplified vector gen-

ome junctions [6,7�], by introduction of adaptor

sequences on the basis of phage Mu transposition [8�]or by sonication of the genome [9�].

The recent implementation of high-throughput sequen-

cing exponentially increased the number of available

retroviral IS. However, as now millions of single

sequences can be produced in a few days, sophisticated

data analysis tools are indispensable. Recently, we and

others published bioinformatical tools both for automatic

processing of raw sequences [10,11] and downstream data

analysis [12,13].

Integration site selection by retroviral vectorsFor many years structural insights into the complex of IN

with the host DNA were limited. Recently, the structure

of the prototype foamy virus (PFV) IN was resolved with

high resolution by X-ray crystallography [14�]. The inta-

some — the complex of viral DNA with IN — appears to

be a dimer-of-dimers of IN, where only one subunit of the

dimer binds to the viral DNA end. During strand transfer

reaction, severe bending of (chromosomal) target DNA

allows the active sites of the intasome to access and cleave

the target phosphodiester bonds [15��]. Consequently,

PFV favors integration into genomic sequences harboring

central, flexible pyrimidine-purine dinucleotides. As

expected, base-specific interactions of IN with target

DNA are few. The structure of PFV-IN allows modeling

the structure of other retroviral IN, like HIV [16]. This

modeling can serve as a starting point for the rational

design of sequence specific retroviral IN [17]. Progress in

structural biology of retroviral IN beyond this brief over-

view was reviewed in detail recently [18].

Retroviral IN must confer its action on two types of DNA,

the viral LTR ends and the chromosomal target DNA.

Suicidal autointegration is prevented by strong uracilation

of the retroviral genome (>5%) [19]. In contrast to the

sequence-specific recognition of viral DNA by IN [20],

selectivity on the target DNA is more promiscuous, as

strong sequence specificity would be disadvantageous by

limiting the frequency of potential integration loci. How-

ever, weak palindromic consensus sequences are com-

monly found at retroviral IS [21,22]. Retroviruses show

genus-specific preferences for integration. Since the first

draft of the human genome sequence, numerous IS

studies have revealed distinct patterns for different retro-

viruses. Murine leukemia viruses (MLV) and derived

vectors from the gammaretrovirus genus show a strong

preference for integration close to transcription start sites

(TSS) and CpG islands [23–25]. In contrast, lentiviral

vectors prefer integration inside transcription units of

actively transcribed genes [26] that cluster in mega-

base-wide chromosomal regions [27��].


Alpha-retroviral vectors show a largely random and

uniform integration pattern [25,28]. Similar, rather close

to random IS distribution has been shown for PFV [29,30].

However, IS analysis uncovers only endpoint scenarios.

Efficient IS analysis in freshly transduced cells is ham-

pered by the presence of nonintegrated episomal vector

forms. Thus, different IS patterns reflect integrations

persisting until time of analysis, which is not necessarily

congruent with the initial IS preference. For example,

potential deleterious (and apoptotic) integration events

can not be detected. Furthermore, a direct comparison of

IS obtained from pretransplantation samples and later

time points from patient samples in hematopoietic stem

cell gene therapy is limited, as only a minor fraction of the

initially transduced heterogenic CD34+ enriched cells

will engraft and show self-renewal capacity.

The causal mechanisms underlying preferential integ-

ration into particular regions of the genome are still not

dissolved. Substituting IN of a HIV-1 derived lentiviral

vector by MLV IN changed the characteristic lentiviral

integration pattern into a gammaretroviral-like integ-

ration pattern, indicating that the viral IN plays a major

role in target site selection [24]. However, on the genome

level host factors are thought to confer target site selec-

tion and interactions of the viral IN with cellular proteins

(see below) as well as influence of transcriptional activity

[23,31] and chromatin status [32] are likely further deci-

sive. As such, cell type specific integration pattern have

been reported for both gammaretro viral and lentiviral

vector systems [33�,34,35]. For gammaretroviral vectors a

preferential integration into genomic regions enriched

with cell-type specific subsets of transcription factor

binding sites has been observed. IN and LTR enhancer

appeared to determine the tethering of retroviral pre-

integration complexes to transcriptionally active regulat-

ory regions [31]. With regards to HIV-1 based vectors,

lens epithelium-derived growth factor (Psip1/Ledgf/p75)

is the best studied IN interacting protein tethering vector

integration into actively transcribed genes. Downregula-

tion of Psip1/Ledgf/p75 results in decreased preferential

integration into transcription units [36,37]. Similarly, we

recently showed close to random integration into genes in

rodent postmitotic tissue, that express Psip1/Ledgf/p75 to

much lower levels compared to dividing cells [33�].Several other studies recently showed that lentiviral

integration can be retargeted into heterochromatin by

replacing the Psip1/Ledgf/p75 chromatin interaction

domain [38–40]. Another study revealed that knockdown

of nuclear pore proteins Transportin-3 and RanBP2

decreased targeting of HIV into gene dense regions,

suggesting that the nuclear pore may be involved in

trafficking HIV to preferred IS [41].

Influence of proviral DNA on the host genomeIntegration of a foreign DNA is per se a mutagenic

event and can lead to malignant transformation of cells


594 Immunogenetics and transplantation

harboring retroviral integrants. Indeed, retroviruses were

discovered by their ability to transform cells and have

been extensively used to identify oncogenes in the gen-

ome. Genes identified by such mutagenesis studies are

listed in the retroviral tagged cancer gene database

RTCGD [42]. In clinical gene therapy trials using gam-

maretroviral vectors insertional mutagenesis resulted in

severe side effects like clonal expansion of gene corrected

cells or T-cell leukemia due to upregulation of proto-

oncogenes [43–46].

Insertional mutagenesis may be linked to preferential

integration of gammaretroviral vectors into gene regu-

latory elements. In contrast, other gene therapy trials

using similar gammaretroviral vectors to treat CGD [47]

and adenosine deaminase (ADA-) SCID [48,49] showed

no severe side effects by now. Lentiviral integration

profiles may be beneficial in terms of avoiding insertional

mutagenesis. Recently, HIV-1 based self-inactivating

(SIN) lentiviral vectors have been successfully used to

treat X-linked adrenoleukodystrophy [50] and beta-

thalassaemia [51].

However, integration into transcribed genes has the

potential to deregulate gene expression on posttranscrip-

tional level. Thus, characteristic targeting of transcription

units by lentiviral vectors possesses also a safety risk.

Indeed, in the beta-thalassaemia trial a clonal expansion

has been attributed to vector integration induced expres-

sion of a truncated HMGA2 transcript [51]. Recently, two

papers reported an unexpected high level of aberrantly

spliced chimeric transcripts in targeted genes [52��,53��].SIN configuration of the vector LTR sharply reduced the

levels of aberrant splicing products compared to full LTR

carrying vectors [52]. Another study recently reported an

acute B-lymphoblastic leukemia (B-ALL) caused by

insertional gene inactivation of a tumor suppressor gene

by the lentiviral vector [54�]. Also stable integration of

both gammaretroviral and lentiviral vectors can signifi-

cantly alter the nuclear chromatin organization of an

endogenous locus [55].

Application of nonintegrating retroviralvectorsIntegration deficient vectors are attractive alternatives for

gene transfer into postmitotic cells as the risk of inser-

tional mutagenesis is greatly reduced. Besides integ-

ration, IN exhibits non-catalytic functions, which are

essential for viral replication. Thus, IN mutants are sub-

divided in two classes, either affecting the DDE catalytic

triad (Class I) or mutations impairing other steps of the

retroviral life cycle (Class II). Integrase defective lenti-

viral vectors (IDLV) have been used successfully for long-

term expression of transgenes [56] or inducing RNA

interference by short hairpin RNAs (shRNAs) in non-

dividing cells [57]. In actively dividing cells, IDLV have

been used for short-term expression of their cargo, for


example, expression of zinc finger nucleases (ZFN) to

confer targeted genome modification [58,59] or as hybrid

vectors for non-viral systems benefiting from the broad

tropism of lentiviral vectors [60,61]. Although canonical

IN-mediated integration is abolished in these vectors,

IDLV are still able to integrate their genome into the host

genome, likely by non-homologous end-joining into cel-

lular double strand breaks — similar to what was

described for adeno-associated viral vectors. Neverthe-

less, frequency of integration is strongly reduced com-

pared to integrating lentiviral vectors and IS show random

integration throughout the host genome [58,62�].

ConclusionsThe incidence of severe side effects in clinical gene

therapy trials has fostered the necessity of performing

sophisticated IS analysis studies and to continue devel-

oping improved retroviral vector. IS analysis uncovered

specific integration preferences for various retroviral

genera. Gammaretroviruses favor gene regulatory

regions of actively transcribed genes. Lentiviruses exhi-

bit a potentially safer integration profile. However lenti-

viral vector induced clonal expansion of gene modified

cells in patients and even onset of leukemia in mouse

models by the disruption of expressed genes have been

observed. Also high levels of aberrantly spliced fusion

transcripts are a concern related to lentiviral vector

integration profile. In postmitotic tissue IDLV are an

attractive option as they almost exclusively persist as

episomes in the nucleus and therefore do not interfere

directly with the host genome. With respect to target site

selection, alpharetroviral and foamyviral vectors may

even be more beneficial in reducing the risk for inser-

tional mutagenesis as they show close to random integ-

ration. However, probably most promising future gene

therapy approaches will endeavor targeting the gene

transfer vector into a specific safe harbor position in

the genome. The recent resolution of the PFV IN will

reinforce attempts to develop site-specific IN versions.

Designer nucleases like ZFN, transcription activator-

like effector nucleases (TALEN) or meganucleases have

the advantage to modify the host genome in a sequence-

specific manner. This not only enables us to insert

exclusively a transgene sequence into the host genome

but also paves the way to repair nonfunctional genes

directly at the endogenous position without perturbing

the remaining genome. To achieve the latter, highly

specific nucleases have to be developed, that guarantee

specific DNA cleavage and efficient integration of the

desired genetic information into the host cell.

AcknowledgementsThe authors are grateful to all members of our lab and our collaborationpartners who participated in this work. This research has been funded inpart by the Deutsche Forschungsgemeinschaft (SPP1230, grant of theTumor Center Heidelberg/Mannheim), by the Bundesministerium furBildung und Forschung (iGene), by the VIth + VIIth Framework Programsof the European Commission (EC, European Network for the



Advancement of Clinical Gene Transfer and Therapy (CLINIGENE) andPersisting Transgenesis (PERSIST) and by the Initiative and NetworkingFund of the Helmholtz Association within the Helmholtz Alliance onImmunotherapy of Cancer to C.v.K. and M.S.

References and recommended readingPapers of particular interest, published within the period of review,have been highlighted as:

� of special interest

�� of outstanding interest

1. Nowotny M: Retroviral integrase superfamily: the structuralperspective. EMBO Rep 2009, 10:144-151.

2. Delelis O, Carayon K, Saib A, Deprez E, Mouscadet JF: Integraseand integration: biochemical activities of HIV-1 integrase.Retrovirology 2008, 5:114.

3. Schmidt M, Schwarzwaelder K, Bartholomae C, Zaoui K, Ball C,Pilz I, Braun S, Glimm H, von Kalle C: High-resolution insertion-site analysis by linear amplification-mediated PCR(LAM-PCR). Nat Methods 2007, 4:1051-1057.

4. Mueller PR, Wold B: In vivo footprinting of a muscle specificenhancer by ligation mediated PCR. Science 1989, 246:780-786.

5. Pfeifer GP, Steigerwald SD, Mueller PR, Wold B, Riggs AD:Genomic sequencing and methylation analysis by ligationmediated PCR. Science 1989, 246:810-813.

6. Gabriel R, Eckenberg R, Paruzynski A, Bartholomae CC,Nowrouzi A, Arens A, Howe SJ, Recchia A, Cattoglio C, Wang Wet al.: Comprehensive genomic access to vector integration inclinical gene therapy. Nat Med 2009, 15:1431-1436.

7.�

Paruzynski A, Arens A, Gabriel R, Bartholomae CC, Scholz S,Wang W, Wolf S, Glimm H, Schmidt M, von Kalle C: Genome-widehigh-throughput integrome analyses by nrLAM-PCR and next-generation sequencing. Nat Protocols 2010, 5:1379-1395.

8.�

Brady T, Roth SL, Malani N, Wang GP, Berry CC, Leboulch P,Hacein-Bey-Abina S, Cavazzana-Calvo M, Papapetrou EP,Sadelain M et al.: A method to sequence and quantify DNAintegration for monitoring outcome in gene therapy. NucleicAcids Res 2011, 39:e72.

9.�

Gillet NA, Malani N, Melamed A, Gormley N, Carter R, Bentley D,Berry C, Bushman FD, Taylor GP, Bangham CR: The hostgenomic environment of the provirus determines theabundance of HTLV-1-infected T-cell clones. Blood 2011,117:3113-3122.

These three papers desribe unbiased methods that allow genome-widedetection of retroviral integration sites.

10. Arens A, Appelt JU, Bartholomae CC, Gabriel R, Paruzynski A,Gustafson D, Cartier N, Aubourg P, Deichmann A, Glimm H et al.:Bioinformatic clonality analysis of next-generationsequencing-derived viral vector integration sites. Hum GeneTher Methods 2012, 23:111-118.

11. Hawkins TB, Dantzer J, Peters B, Dinauer M, Mockaitis K,Mooney S, Cornetta K: Identifying viral integration sites usingSeqMap 2.0. Bioinformatics 2011, 27:720-722.

12. Abel U, Deichmann A, Nowrouzi A, Gabriel R, Bartholomae CC,Glimm H, von Kalle C, Schmidt M: Analyzing the number ofcommon integration sites of viral vectors — new methods andcomputer programs. PLoS One 2011, 6:e24247.

13. Presson AP, Kim N, Xiaofei Y, Chen IS, Kim S: Methodology andsoftware to detect viral integration site hot-spots. BMCBioinformatics 2011, 12:367.

14.�

Hare S, Gupta SS, Valkov E, Engelman A, Cherepanov P:Retroviral intasome assembly and inhibition of DNA strandtransfer. Nature 2010, 464:232-236.

This study provided for the first time the X-ray structure of a full-lengthretroviral integrase in complex with viral DNA. The authors present thearchitectural basis of the integration mechanism of prototyp foamyvirus.

15.��

Maertens GN, Hare S, Cherepanov P: The mechanism ofretroviral integration from X-ray structures of its keyintermediates. Nature 2010, 468:326-329.


This study showed for the first time the X-ray structure of a retroviralintegrase in complex with its target DNA, both before and after strandtransfer. The authors resolved the structural basis of retroviral integrationand thus provided a starting point for the rational design for target sitespecific retroviral integrases.

16. Krishnan L, Li X, Naraharisetty HL, Hare S, Cherepanov P,Engelman A: Structure-based modeling of the functional HIV-1intasome and its inhibition. Proc Natl Acad Sci U S A 2010,107:15910-15915.

17. Lim KI, Klimczak R, Yu JH, Schaffer DV: Specific insertions ofzinc finger domains into Gag-Pol yield engineered retroviralvectors with selective integration properties. Proc Natl AcadSci U S A 2010, 107:12475-12480.

18. Engelman A, Cherepanov P: The structural biology of HIV-1:mechanistic and therapeutic insights. Nat Rev Microbiol 2012,10:279-290.

19. Yan N, O‘Day E, Wheeler LA, Engelman A, Lieberman J: HIV DNAis heavily uracilated, which protects it from autointegration.Proc Natl Acad Sci U S A 2011, 108:9244-9249.

20. Brin E, Leis J: HIV-1 integrase interaction with U3 and U5terminal sequences in vitro defined using substrates withrandom sequences. J Biol Chem 2002, 277:18357-18364.

21. Holman AG, Coffin JM: Symmetrical base preferencessurrounding HIV-1, avian sarcoma/leukosis virus, and murineleukemia virus integration sites. Proc Natl Acad Sci U S A 2005,102:6103-6107.

22. Wu X, Li Y, Crise B, Burgess SM, Munroe DJ: Weak palindromicconsensus sequences are a common feature found at theintegration target sites of many retroviruses. J Virol 2005,79:5211-5214.

23. Wu X, Li Y, Crise B, Burgess SM: Transcription start regions inthe human genome are favored targets for MLV integration.Science 2003, 300:1749-1751.

24. Lewinski MK, Yamashita M, Emerman M, Ciuffi A, Marshall H,Crawford G, Collins F, Shinn P, Leipzig J, Hannenhalli S et al.:Retroviral DNA integration: viral and cellular determinants oftarget-site selection. PLoS Pathog 2006, 2:e60.

25. Mitchell RS, Beitzel BF, Schroder AR, Shinn P, Chen H, Berry CC,Ecker JR, Bushman FD: Retroviral DNA integration: ASLV, HIV,and MLV show distinct target site preferences. PLoS Biol 2004,2:E234.

26. Schroeder AR, Shinn P, Chen H, Berry C, Ecker JR, Bushman F:HIV-1 integration in the human genome favors active genesand local hotspots. Cell 2002, 110:521-529.

27.��

Biffi A, Bartolomae CC, Cesana D, Cartier N, Aubourg P,Ranzani M, Cesani M, Benedicenti F, Plati T, Rubagotti E et al.:Lentiviral vector common integration sites in preclinicalmodels and a clinical trial reflect a benign integration bias andnot oncogenic selection. Blood 2011, 117:5332-5339.

This study showed that common integration site detected in a lentiviralgene therapy trial results from preferential vector integration into mega-base-wide chromosomal regions rather than by vector induced clonalselection.

28. Suerth JD, Maetzig T, Brugman MH, Heinz N, Appelt JU,Kaufmann KB, Schmidt M, Grez M, Modlich U, Baum C et al.:Alpharetroviral self-inactivating vectors: long-term transgeneexpression in murine hematopoietic cells and lowgenotoxicity. Mol Ther 2012, 20:1022-1032.

29. Nowrouzi A, Dittrich M, Klanke C, Heinkelein M, Rammling M,Dandekar T, von Kalle C, Rethwilm A: Genome-wide mapping offoamy virus vector integrations into a human cell line. J GenVirol 2006, 87:1339-1347.

30. Trobridge GD, Miller DG, Jacobs MA, Allen JM, Kiem HP, Kaul R,Russell DW: Foamy virus vector integration sites in normalhuman cells. Proc Natl Acad Sci U S A 2006, 103:1498-1503.

31. Felice B, Cattoglio C, Cittaro D, Testa A, Miccio A, Ferrari G, Luzi L,Recchia A, Mavilio F: Transcription factor binding sites aregenetic determinants of retroviral integration in the humangenome. PLoS One 2009, 4:e4571.


596 Immunogenetics and transplantation

32. Ambrosi A, Glad IK, Pellin D, Cattoglio C, Mavilio F, Di Serio C,Frigessi A: Estimated comparative integration hotspotsidentify different behaviors of retroviral gene transfer vectors.PLoS Comput Biol 2011, 7:e1002292.

33.�

Bartholomae CC, Arens A, Balaggan KS, Yanez-Munoz RJ,Montini E, Howe SJ, Paruzynski A, Korn B, Appelt JU, Macneil Aet al.: Lentiviral vector integration profiles differ in rodentpostmitotic tissues. Mol Ther 2011, 19:703-710.

This study showed that lentiviral vectors exhibit a close to randomintegration profile in rodent postmitotic cells compared to the preferentialintegration into genes in actively deviding cells. The results suggest thatpostmititic tissue might be a safer target for integrating vectors.

34. Brady T, Agosto LM, Malani N, Berry CC, O‘Doherty U,Bushman F: HIV integration site distributions in resting andactivated CD4+ T cells infected in culture. AIDS 2009,23:1461-1471.

35. Biasco L, Ambrosi A, Pellin D, Bartholomae C, Brigida I,Roncarolo MG, Di Serio C, von Kalle C, Schmidt M, Aiuti A:Integration profile of retroviral vector in gene therapy treatedpatients is cell-specific according to gene expression andchromatin conformation of target cell. EMBO Mol Med 2011,3:89-101.

36. Llano M, Vanegas M, Fregoso O, Saenz D, Chung S, Peretz M,Poeschla EM: LEDGF/p75 determines cellular trafficking ofdiverse lentiviral but not murine oncoretroviral integraseproteins and is a component of functional lentiviralpreintegration complexes. J Virol 2004, 78:9524-9537.

37. Ciuffi A, Llano M, Poeschla E, Hoffmann C, Leipzig J, Shinn P,Ecker JR, Bushman F: A role for LEDGF/p75 in targeting HIVDNA integration. Nat Med 2005, 11:1287-1289.

38. Gijsbers R, Ronen K, Vets S, Malani N, De Rijck J, McNeely M,Bushman FD, Debyser Z: LEDGF hybrids efficiently retargetlentiviral integration into heterochromatin. Mol Ther 2010,18:552-560.

39. Silvers RM, Smith JA, Schowalter M, Litwin S, Liang Z, Geary K,Daniel R: Modification of integration site preferences of anHIV-1-based vector by expression of a novel synthetic protein.Hum Gene Ther 2010, 21:337-349.

40. Ferris AL, Wu X, Hughes CM, Stewart C, Smith SJ, Milne TA,Wang GG, Shun MC, Allis CD, Engelman A et al.: Lens epithelium-derived growth factor fusion proteins redirect HIV-1 DNAintegration. Proc Natl Acad Sci U S A 2010, 107:3135-3140.

41. Ocwieja KE, Brady TL, Ronen K, Huegel A, Roth SL, Schaller T,James LC, Towers GJ, Young JA, Chanda SK et al.: HIVintegration targeting: a pathway involving Transportin-3 andthe nuclear pore protein RanBP2. PLoS Pathog 2011,7:e1001313.

42. Akagi K, Suzuki T, Stephens RM, Jenkins NA, Copeland NG:RTCGD: retroviral tagged cancer gene database. Nucleic AcidsRes 2004, 32:D523-D527.

43. Hacein-Bey-Abina S, Von Kalle C, Schmidt M, McCormack MP,Wulffraat N, Leboulch P, Lim A, Osborne CS, Pawliuk R, Morillon Eet al.: LMO2-associated clonal T cell proliferation in twopatients after gene therapy for SCID-X1. Science 2003,302:415-419.

44. Howe SJ, Mansour MR, Schwarzwaelder K, Bartholomae C,Hubank M, Kempski H, Brugman MH, Pike-Overzet K,Chatters SJ, de Ridder D et al.: Insertional mutagenesiscombined with acquired somatic mutations causesleukemogenesis following gene therapy of SCID-X1 patients. JClin Invest 2008, 118:3143-3150.

45. Avedillo Diez I, Zychlinski D, Coci EG, Galla M, Modlich U,Dewey RA, Schwarzer A, Maetzig T, Mpofu N, Jaeckel E et al.:Development of novel efficient SIN vectors with improvedsafety features for Wiskott-Aldrich syndrome stem cell basedgene therapy. Mol Pharm 2011, 8:1525-1537.

46. Stein S, Ott MG, Schultze-Strasser S, Jauch A, Burwinkel B,Kinner A, Schmidt M, Kramer A, Schwable J, Glimm H et al.:Genomic instability and myelodysplasia with monosomy 7consequent to EVI1 activation after gene therapy for chronicgranulomatous disease. Nat Med 2010, 16:198-204.


47. Kang EM, Choi U, Theobald N, Linton G, Long Priel DA, Kuhns D,Malech HL: Retrovirus gene therapy for X-linked chronicgranulomatous disease can achieve stable long-termcorrection of oxidase activity in peripheral blood neutrophils.Blood 2010, 115:783-791.

48. Gaspar HB, Cooray S, Gilmour KC, Parsley KL, Zhang F, Adams S,Bjorkegren E, Bayford J, Brown L, Davies EG et al.:Hematopoietic stem cell gene therapy for adenosinedeaminase-deficient severe combined immunodeficiencyleads to long-term immunological recovery and metaboliccorrection. Sci Transl Med 2011, 3:97ra80.

49. Ferrua F, Brigida I, Aiuti A: Update on gene therapy foradenosine deaminase-deficient severe combinedimmunodeficiency. Curr Opin Allergy Clin Immunol 2010,10:551-556.

50. Cartier N, Hacein-Bey-Abina S, Bartholomae CC, Veres G,Schmidt M, Kutschera I, Vidaud M, Abel U, Dal-Cortivo L,Caccavelli L et al.: Hematopoietic stem cell gene therapy with alentiviral vector in X-linked adrenoleukodystrophy. Science2009, 326:818-823.

51. Cavazzana-Calvo M, Payen E, Negre O, Wang G, Hehir K,Fusil F, Down J, Denaro M, Brady T, Westerman K et al.:Transfusion independence and HMGA2 activation after genetherapy of human beta-thalassaemia. Nature 2010,467:318-322.

52.��

Cesana D, Sgualdino J, Rudilosso L, Merella S, Naldini L,Montini E: Whole transcriptome characterization of aberrantsplicing events induced by lentiviral vector integrations. J ClinInvest 2012, 122:1667-1676.

53.��

Moiani A, Paleari Y, Sartori D, Mezzadra R, Miccio A, Cattoglio C,Cocchiarella F, Lidonnici MR, Ferrari G, Mavilio F: Lentiviralvector integration in the human genome induces alternativesplicing and generates aberrant transcripts. J Clin Invest 2012,122:1653-1666.

These two papers reported unexpectedly high levels of aberrantly splicedchimeric transcripts consisting of sequences belonging to the genetransfer vector and to host genes. Few cryptic splice sites have beenidentified as the major cause for this phenomenon. Removal ofthese splice sites in the vector sequence may aid in developing saferlentiviral gene transfer vectors with reduced risk for posttranscriptionalgenotoxicity.

54.�

Heckl D, Schwarzer A, Haemmerle R, Steinemann D, Rudolph C,Skawran B, Knoess S, Krause J, Li Z, Schlegelberger B et al.:Lentiviral vector induced insertional haploinsufficiency of Ebf1causes murine leukemia. Mol Ther 2012.

This study observed onset of acute B-lymphoblastic leukemia in onemouse due to insertional gene inactivation.

55. Nagel J, Gross B, Meggendorfer M, Preiss C, Grez M, Brack-Werner R, Dietzel S: Stably integrated and expressed retroviralsequences can influence nuclear location and chromatincondensation of the integration locus. Chromosoma 2012,121:353-367.

56. Yanez-Munoz RJ, Balaggan KS, MacNeil A, Howe SJ, Schmidt M,Smith AJ, Buch P, MacLaren RE, Anderson PN, Barker SE et al.:Effective gene therapy with nonintegrating lentiviral vectors.Nat Med 2006, 12:348-353.

57. Hutson TH, Foster E, Dawes JM, Hindges R, Yanez-Munoz RJ,Moon LD: Lentiviral vectors encoding shRNAs efficientlytransduce and knockdown LINGO-1 but induce an interferonresponse and cytotoxicity in CNS neurons. J Gene Med 2012,14:299-315.

58. Gabriel R, Lombardo A, Arens A, Miller JC, Genovese P,Kaeppel C, Nowrouzi A, Bartholomae CC, Wang J, Friedman Get al.: An unbiased genome-wide analysis of zinc-fingernuclease specificity. Nat Biotechnol 2011, 29:816-823.

59. Lombardo A, Genovese P, Beausejour CM, Colleoni S, Lee YL,Kim KA, Ando D, Urnov FD, Galli C, Gregory PD et al.: Geneediting in human stem cells using zinc finger nucleases andintegrase-defective lentiviral vector delivery. Nat Biotechnol2007, 25:1298-1306.

60. Staunstrup NH, Moldt B, Mates L, Villesen P, Jakobsen M, Ivics Z,Izsvak Z, Mikkelsen JG: Hybrid lentivirus-transposon vectors



with a random integration profile in human cells. Mol Ther 2009,17:1205-1214.

61. Vink CA, Gaspar HB, Gabriel R, Schmidt M, McIvor RS,Thrasher AJ, Qasim W: Sleeping beauty transposition fromnonintegrating lentivirus. Mol Ther 2009, 17:1197-1204.

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Matrai J, Cantore A, Bartholomae CC, Annoni A, Wang W,Acosta-Sanchez A, Samara-Kuko E, De Waele L, Ma L,


Genovese P et al.: Hepatocyte-targeted expression byintegrase-defective lentiviral vectors induces antigen-specifictolerance in mice with low genotoxic risk. Hepatology 2011,53:1696-1707.

This study showed that rare integration events of integrase-defectivelentiviral vectors are distributed randomly in the genome and are notmediated by residual viral integrase activity.


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Integration of retroviral vectors