Gene therapy clinical trials by delivery method

Clinical trials by delivery method (%)

GammaretrovirusLentivirus

AdenovirusAAV

Herpes simplex virusPoxvirus

Other virusesLipofection

Naked/Plasmid DNARNA transfer

BacteriaOthers/not classified

0 5 10 15 20 25

3,60,81,4

18,67,1

1,712,0

3,34,4

24,41,4

21,2

Giacca, M. 2010. Gene Therapy. Springer

68.5%

Virus Vector

TRTR

promoter

LTR

LTRvpr

SD SAΨ RRE

promoterHIV -1

LTRLTR

gag polvif

vpr

tatrevenv

nef

vpu

1 kb

ΨRRE

Adenovirus

E1A

1 kb

E1B L1 L2 L3 L4 L5

E3

E4E2AE2B

AAV

rep cap

ITRITRp5 p19 p46

polyadenylation site

1 kb

Murine/avian retroviruses

1 kb

gag polenv

LTR

LTRΨ

SD SA

Ψ LTRLTR therapeutic gene

promoter (?)therapeutic gene(double copy vectors)

SD SA

L1 L2 L3 L4 L5

E3

E4E2AE2B

promoter

therapeutic gene

therapeutic gene

therapeutic gene

Hematopoiesis

The whole hematopoietic system can be reconstituted by a single HSC

Enrichment of CD34+ CD38- hematopoietic precursors from the bone marrow and from peripheral blood after mobilization

Principali difetti molecolare che portano allo sviluppo di SCID. AR: autosomica recessiva; X-L: legata al cromosoma X

Meccanismo Gene mutato Ereditarieta’ Cellule affette

Morte prematura delle cellule

ADA AR T, B, NK

Difetto nella sopravvivenza dovuto alla mancanza di segnali attivatori da parte di citochine

catena comune γ (cγ) X-L T, NK

JAK-3 AR T, NK

1L7RA AR T

Difetto nel riarrangiamento V(D)J

RAG1 o RAG2 AR T, B

Artemis AR T, B

Difetto nella segnalazione da parte del pre-TCR o del TCR

CD3 δ, ζ, ε AR T

CD45 AR T

La convenzione classica della terapia genica non corregge ma aggiunge una copia sana del gene mutato

Principali successi ad oggi ottenuti per le malattie AR

Nuove prospettive con gene editing

HSCLymphoidprogenitor

PrecursorNK/T cell

NK cell

DN1 DN2 DN3 ISP DP

SP

SP CD4+T cell

CD8+T cell

Pro-Bcell

Pre-B-Icell

Large pre-B-IIcell

Small pre-B-IIcell

ImmatureB cell

MatureB cell

Plasmacell

Bone marrow

Thymus

Blood

RAG1RAG2

ArtemisDNA ligase IV

RAG1RAG2

ArtemisDNA ligase IV

CD3ε CD3δ

CD45CD3γCD3ζγchain

JAK3IL7RA

γchainJAK3

ADA

Defects leading to the development of severe combined immunodeficiency

(SCID)

The Nobel Prize in Physiology or Medicine 1975

The Nobel Prize in Physiology or Medicine 1975 was awarded jointly to David Baltimore, Renato Dulbecco and Howard Martin Temin "for their discoveries concerning the interaction between tumour viruses and the genetic material of the cell."

Adsorption

Fusion

Uncoating

Transport to the nuclues

Integration

Transcription

SplicingNuclear export of viral mRNAs

Translation of Env polyprotein

Translation of Gag and Gag-Pol polyproteins

Assembly of virions

Budding and polyprotien maturation

Maturation of TM and SU and transport to the cell surface

Reverse transcription

Retrovirus life cycle

Giacca, M. 2010. Gene Therapy. Springer

Taxonomy of the Retroviridae familySubfamily Genus Former classifications Main species Prototype viruses

Orthoretrovirinae

Alpharetrovirus Avian type C retroviruses; Avian sarcoma/leukosis viruses (ASLV)

Avian leukosis virus ALVRous sarcoma virus RSV

Betaretrovirus Mammalian type B retroviruses; Type D retroviruses

Mouse mammary tumor virus MMTVMason-Pfizer monkey virus MPMV

Gammaretrovirus Mammalian type C retroviruses

Murine leukemia virus Abelson-MLV, Friend-MLV, Moloney-MLV

Feline leukemia virus FeLVGibbon ape leukemia virus GaLVHarvey murine sarcoma virus Ha-MSVMoloney murine sarcoma virus Mo-MSVSimian sarcoma virus SSVReticuloendotheliosis virus REV-A, REV-T

Deltaretrovirus BLV-HLTV group retroviruses

Bovine leukemia virus BLV

Primate T-lymphotropic viruses (human and simian)

HTLV-1, STLV-1, HTLV-2, STLV-2, STLV-3

Epsilonretrovirus Fish retroviruses Walleye dermal sarcoma virus WDSV

Lentivirus

Bovine immunodeficiency virus BIVEquine infectious anemia virus EIAVFeline immunodeficiency virus FIV-O, FIV-PCaprine arthritis encephalitis virus CAEV

Visna/Maedi virus VISNAHuman immunodeficiency virus 1 and 2 HIV-1, HIV-2

Simian immunodeficiency virus SIV-agm.155, SIV-cpz, SIV-mac

Spumaretrovirinae Spumavirus

Simian foamy virus

SFVmac (SFV-1 and SFV-2), SFVagm (SFV-3), SFVcpz and SFVcpz(hu)

Bovine foamy virus BFVEquine foamy virus EFVFeline foamy virus FFVHuman foamy virus HFV or HSRV

Genetic organization of retroviruses

Oncoretrovirus

Lentivirus

Spumavirus

Complex retroviruses

Genomic organization of retroviruses

gag pol

LTRLTR

HFVenv Bel 2

Bel 3

Bel 1(Taf)

gag polvif vpr rev

tat

env

nef

vpxLTRLTR

HIV-2, SIV

gag polvif

vpr

tatrev

env

nef

vpuLTRLTR

HIV-1

gag pol taxrex

envLTRLTR

HTLV I,II

MMTV3'ORFgag pol

env

LTRLTR

RSVgag pol

env

LTRLTR

src

MLVgag pol

env

LTRLTRCommon features of retroviruses

Genomic organization of retroviruses

gag pol

LTRLTR

HFVenv Bel 2

Bel 3

Bel 1(Taf)

gag polvif vpr rev

tat

env

nef

vpxLTRLTR

HIV-2, SIV

gag polvif

vpr

tatrev

env

nef

vpuLTRLTR

HIV-1

gag pol taxrex

envLTRLTR

HTLV I,II

MMTV3'ORFgag pol

env

LTRLTR

RSVgag pol

env

LTRLTR

src

MLVgag pol

env

LTRLTR

They all contain LTRs (400-700 nt), which form in the integrated pro-virus

Viral particles contain mRNA

They all contain gag, pol and env genes

The construction of a src-specific DNA probe.

A version of the src gene carried by RSV is also present in uninfected cells

Structure of the RSV genomeThe discovery of proto-oncogenes: a version of the src gene carried by RSV is also present in uninfected cells

Parental /helper virus Retrovirus Acronym v-onc

Rous sarcoma virus RSV src

Avian leukosis virus (ALV)

Avian myeloblastosis virus AMV mybAvian erythroblastosis virus AEV erbA, BAvian myelocytomatosis virus 29 AMCV-29 myc

Y73 sarcoma virus Y73SV yesAvian sarcoma virus 17 ASV-17 jun

Moloney-Murine leukemia virus (Mo-MLV)

Abelson murine leukemia virus Ab-MLV abl

Harvey murine sarcoma virus Ha-MSV rasMoloney murine sarcoma virus Mo-MSV mosFinkel-Biskis-Jinkins murine sarcoma virus FBJ-MSV fos

Feline leukemia virus (FeLV)Snyder-Theilen feline sarcoma virus ST-FeSV

fesGardner-Arnstein feline sarcoma virus GA-FeSVSusan McDonough feline sarcoma virus SM-FeSV fms

Hardy-Zuckerman 4 feline sarcoma virus

HZ4-FeSV kit

Simian sarcoma virus (SSV) Woolly monkey sarcoma virus WMSV sis

Examples of retroviruses carrying viral oncogenes (v-onc)

Slowly transforming retroviruses activate protooncogenesby inserting their genomes adjacent to these cellular genes

Some retroviruses naturally carry oncogenes

Insertional mutagenesis

ALV/ lack acquired oncogenesB-call lymphomas induced by ALV

HTLV-I/ tax (transcription activator)

Slowly transforming retroviruses activate protooncogenesby inserting their genomes adjacent to these cellular genes

Some retroviruses naturally carry oncogenes

Insertional mutagenesis

ALV/ lack acquired oncogenesB-call lymphomas induced by ALV

HTLV-I/ tax (transcription activator)

SUTM

lipid bilayer

MACA

NC

RNA

RT

IN

PR

Retrovirus virion

Reverse transcription (RT)

Transcription (RNA Pol II)

gag pol

env

LTR

U3 R U5

LTR

U3 R U5

Integrated provirus (DNA)

5’ CAP

R U5 U3 R

Viral genome (mRNA)

AAA 3’

PBS PPT

Splicing

5’ CAP

R U5 U3 R

AAA 3’env Env mRNA

5’ CAP

R U5 U3 R

Viral genome and Gag/Pol mRNAAAA 3’

SD SA

gag pol

ψ

Genetic organization of generalized retrovirus

SUTM

lipid bilayer

MACA

NC

RNA

RT

IN

PR

Genetic organization of generalized retrovirus

SUTM

lipid bilayer

MACA

NC

RNA

RT

IN

PR

Trascrizione inversa (RT)

Trascrizione (RNA Pol II)

gag pol

env

LTR

U3 RU5

LTR

U3 RU5Provirus integrato (DNA)

5’ CAP

RU5 U3 RGenoma virale (mRNA)

AAA 3’

PBS PPT

Splicing

5’ CAP

RU5 U3 R

AAA 3’env mRNA per Env

5’ CAP

RU5 U3 R Genoma virale ed mRNA per Gag/PolAAA 3’

SD SA

gag pol

ψ

gene terapeutico

LTR

U3 R

LTR

U3 RU5Vettore retrovirale (DNA)

PBS PPTU5

ψ

SD SA

Elementi genetici dei Retrovirus

La trascrizione parte da LTR del virus integrato

Nel vettore virale rimane:- LTR che serve per la trascrizione,- una regione di gag che serve per incapsidamento - una porzione che produce un tRNA che funziona da primer per la trascrittasi inversa

Packaging of gammaretroviral vectors

Retroviral vector integration results in transgene transcription (no additional particles produced)

Pseudotyping of gammaretroviral vectors

Disease group Disease Defective gene

Severe combined immunodeficiency syndromes (SCID)

SCID-X1 Gamma common (γc) chain of interleukin receptors

ADA-SCID Adenosine deaminase

JAK-3

PNP-SCID Purine-nucleoside phosphorylase (PNP)

Lysosomal storage disorders

Hurler's disease (MPS I) α-L-iduronidase

Hunter's disease (MPS II) Iduronate-2-sulfatase

Gaucher's disease Glucocerebrosidase (β-glucosidase)

Fabry's disease α-galactosidase A

Sly syndrome (MPS VII) β-glucuronidase

Defects of phagocytes Chronic granulomatous disease (CGD) gp91phox, p47phox

Leukocyte adhesion disorder CD18 (β2-integrin)

Other diseases Fanconi anemia, group C FANCC

Monogenic hereditary disorders for which gene therapy clinical trials were conducted by gene transfer into HSCs

Stimuli following

phagocytosis

G protein

NADPH NADP+

O2 O2-

Outside(phagosome)

Cytosol

gp91 p22

Phospholipase C, D, A2

Increase in IP3, DAG, AA concentration

PKC

Other kinases

gp91

p47

p67

Rap1a Rap1a

Rac1p40

p22

P

p47P

p47

P

p47

p67Rac1

p40

P

Activation of phagocyte NAPDH oxidase

X-CGD patient Normal

X-CGD patient after gammaretrovirus-mediated gp91-phox gene transfer

Functional correction of NAPDH activity in myeloid colonies from an X-CGD patient after gene transfer of the

gp91phox cDNA into CD34+ hematopoietic stem cells

Zentilin, L, et al. 1996. Exp. Cell. Res. 225, 257.

Gene therapy of hematopoietic stem cells: Conclusions from clinical trials

Virus-positive cells are detectable in peripheral blood after several years from treatment

Only a very small fraction (0.01-0.1%) of reconstituting HSCs are transduced with the currently available protocols

Quiescent stem cell

binding of retrovirus to specific receptors of the cell surface

reverse transcription and integration into the host cell genome

expression of the transgene in the appropriate cell type

Progenitor cellsMature peripheral blood

cells

Division

Proliferation Differentiation

+ IL-3, IL-6, SCF

Gene therapy of hematopoietic stem cells

HSCLymphoidprogenitor

PrecursorNK/T cell

NK cell

DN1 DN2 DN3 ISP DP

SP

SP CD4+T cell

CD8+T cell

Pro-Bcell

Pre-B-Icell

Large pre-B-IIcell

Small pre-B-IIcell

ImmatureB cell

MatureB cell

Plasmacell

Bone marrow

Thymus

Blood

RAG1RAG2

ArtemisDNA ligase IV

RAG1RAG2

ArtemisDNA ligase IV

CD3ε CD3δ

CD45CD3γCD3ζγchain

JAK3IL7RA

γchainJAK3

ADA

Defects leading to the development of severe combined immunodeficiency

(SCID)

SCID-X1, the bubble boy disease

IL-2

γc

IL2-Rβ

IL2-Rα

JAK3JAK1

IL-15

γc

IL15-Rβ

IL15-Rα

JAK1 JAK3

IL-4

γcIL4-R

JAK1 JAK3

IL-7

γcIL7-Rα

JAK1 JAK3

IL-9

γcIL9-R

JAK1 JAK3

IL-21

γcIL21-R

JAK1 JAK3

Molecular structure of the interleukin receptors

γc gene therapy trial A. Fisher, Paris 2000

Eligibility

SCID-X1 (proven γc gene mutation) Lack of an HLA identical donor

Protocol

Bone marrow harvesting (30-100 ml) CD34+ cell separation (immunomagnetic micro beads) One day pre-activation with SCF, FLT 3L, IL-3 and MGDF Three rounds of infection with the MFG γc vector-containing supernatants in CH-296 fibronectin fragment-coated bags I.V. infusion

Science. 2000. Vol. 288, pp. 669-672

SCID-X1 has been a suitable and attractive setting for the clinical translation of targeted gene correction strategies and adoptive transfer of gene-corrected cells, as cells bearing a functional gamma chain show a positive selective advantage in vivo in the affected patients

Trials of gene therapy for SCID were halted in the United States and France following the report that a three-year-hold patient treated by Alain Fischer in Paris had developed leukemia after being treated with a retroviral vector (ex vivo transduction of bone marrow stem cells).

In October 2002 an advisory committee to the FDA ruled that gene therapy trials of that kind should now continue. However, there must be increased monitoring for adverse events (abnormal activity of certain cells, integration sites), and patients must receive modified informed consent forms to explain the chances of this side effect occurring. “One adverse event, as serious as it is, in the context of the whole field us not enough to put all programs on hold”.

A baby cured of SCID by gene therapy

Using a PCR-based technique, it was discovered that the retroviral vector had inserted into more than 40 sites in the genome of different repopulating cells. In the T-cell clone that grew abnormally, it had inserted in the LMO-2 oncogene, causing increased expression of the gene. Increased activity of the T-cell clone carrying the LMO-2 integration was detected in blood samples taken from the boy as early as 13 months after treatment, well before he showed any clinical symptoms. However, this event was probably not sufficient for leukemia, but a second event was required for cancer to ensue. Another question is the possibility that the boy had a genetic predisposition to leukemia, as there have been two childhood cancers in the family.

December 2002

Insertional mutagenesis

Patient. 1

Patient. 2

LMO2 encodes a LIM domain protein that binds to transcription factors SCL/TAL1, GATA1, GATA2Expressed by haematopoietic progenitors and cells of myeloid lineage, but not in post-thymic T cellsLMO2 is activated in childhood ALL and in other spontaneous human T cell leukaemiasLMO2 is leukaemogenic when overexpressed in transgenic mice

The finding that a retrovirally induced mouse leukaemia contains integrations at both Lmo2 and γc loci provided genetic evidence for cooperativity between LMO2 and γc

Good news for gene therapy

In most gene therapy trials, the transplanted gene is unlikely to be oncogenic and occurrences of insertional mutagenesis will be low, as has been seen in trials conducted during the past several years

Science, 2002

The first report of immune restoration in 2 patients with ADA-deficient SCID These subjects were not treated with PEG-ADA enzyme-replacement therapy, thought to reduce selective advantage of the genetically corrected cellsThese subjects received BM cytoreduction with a moderate dosage of busulphan, which could promote engraftment of the genetically modified cells

1908 Chicken leukosis is caused by a virus (Ellerman and Bang)

1911 Cell-free transmission of sarcoma in chickens (Rous)

1936 Mammary carcinoma in mice caused by a filterable agent (Bittner)

1958 Development of the focus assay for RSV (Temin and Rubin)

1964 Provirus hypothesis (generation of viral DNA copy and integration in cellular genome) (Temin)

1970 Reverse Transcriptase (Temin and Mizutani; Baltimore)

1976 Probe for src oncogene hybridizes with cellular DNA (Stehelin)

1980/82: First human retrovirus (HTLV-I)

1983: HIV-1

Retroviruses: historical introduction

Modern taxonomy of the Retroviridae familySubfamily Genus Former classifications Main species Prototype viruses

Orthoretrovirinae

Alpharetrovirus Avian type C retroviruses; Avian sarcoma/leukosis viruses (ASLV)

Avian leukosis virus ALVRous sarcoma virus RSV

Betaretrovirus Mammalian type B retroviruses; Type D retroviruses

Mouse mammary tumor virus MMTVMason-Pfizer monkey virus MPMV

Gammaretrovirus Mammalian type C retroviruses

Murine leukemia virus Abelson-MLV, Friend-MLV, Moloney-MLV

Feline leukemia virus FeLVGibbon ape leukemia virus GaLVHarvey murine sarcoma virus Ha-MSVMoloney murine sarcoma virus Mo-MSVSimian sarcoma virus SSVReticuloendotheliosis virus REV-A, REV-T

Deltaretrovirus BLV-HLTV group retroviruses

Bovine leukemia virus BLV

Primate T-lymphotropic viruses (human and simian)

HTLV-1, STLV-1, HTLV-2, STLV-2, STLV-3

Epsilonretrovirus Fish retroviruses Walleye dermal sarcoma virus WDSV

Lentivirus

Bovine immunodeficiency virus BIVEquine infectious anemia virus EIAVFeline immunodeficiency virus FIV-O, FIV-PCaprine arthritis encephalitis virus CAEV

Visna/Maedi virus VISNAHuman immunodeficiency virus 1 and 2 HIV-1, HIV-2

Simian immunodeficiency virus SIV-agm.155, SIV-cpz, SIV-mac

Spumaretrovirinae Spumavirus

Simian foamy virus

SFVmac (SFV-1 and SFV-2), SFVagm (SFV-3), SFVcpz and SFVcpz(hu)

Bovine foamy virus BFVEquine foamy virus EFVFeline foamy virus FFVHuman foamy virus HFV or HSRV

Genetic organization of retroviruses

Oncoretrovirus

Lentivirus

Spumavirus

Complex retroviruses

Genomic organization of retroviruses

gag pol

LTRLTR

HFVenv Bel 2

Bel 3

Bel 1(Taf)

gag polvif vpr rev

tat

env

nef

vpxLTRLTR

HIV-2, SIV

gag polvif

vpr

tatrev

env

nef

vpuLTRLTR

HIV-1

gag pol taxrex

envLTRLTR

HTLV I,II

MMTV3'ORFgag pol

env

LTRLTR

RSVgag pol

env

LTRLTR

src

MLVgag pol

env

LTRLTR

9 kb mRNA class

Time after HIV-1 infected cell stimulation (h):

4 kb mRNA class

2 kb mRNA class

1 8 24

LTR gag vif LTRpol env

tat

vprvpunef

rev

1 2 3 4 6D4A4B5

77A7B

64AE4BE5E

2A

Genes

Splicingsites

Exons

mRNAs

Primary transcript

full length, primary mRNA (~ 9 kb)

double-spliced mRNAs (~ 2 kb)

single-spliced mRNAs (~ 4 kb)

genomeGagPol

EnvVifVpuVpr

TatRevNef

RRE/Rev

A7A A7

D5

A4

D3

A2

D2

A1

D1

A5

A3

A4BA4A

A5

D4

HIV-1 genomic organization and transcripts

Export of unspliced viral mRNAs outside the nucleus

gag

pol env

vif

vpr vpu

tat

rev

nef LTR

U3 R U5

PPT

LTR

U3 R

PBS

ψ

SD

U5

RRE

Vettori Lentivirali di prima generazione

SASD

CMV

Poly A

VSV-G

SA

U3 R U5R

PBS

ψ

SD

U5

RRE

gene terapeuticoG E

LTR LTR

SASD

CMV

Poly A

gag

pol

vif

vpr vpu

tat

rev

nef

RRE

ψ

Vettori lentivirali

3 plasmidi

1. segnali regolatori, sito di legame per REV (RRE), promotore e gene terapeutico

2. gag, pol e 6 geni accessori

3. VSV-G (env di HIV lega CD4, espresso essenzialmente in linfociti e macrofagi)

Safety Concerns Specific to Lentiviral vectors

• Recombination during manufacture may generate a replication-competent lentivirus (RCL)

- HIV a known human pathogen - vesicular stomatitis virus (VSV-G) envelope

broadens tropism • Recombination with wild type virus in HIV+ subjects • Lentiviral vector mobilization by wild type virus

RSV rev

Poly A

SASD

CMV

Poly A

VSV-G

SA

U3 RU5R

PBSψ

SD

U5

RRE

RSV gene terapeuticoG E

gagpolSA

RRE

SDCMV

Poly A

E

Vettori lentivirali di terza generazione

ψ

SASDCMV

Poly A

VSV-G

SA

U3 RU5R

PBSψ

SD

U5

RRE

gene terapeuticoG E

Vettori lentivirali di seconda generazione

LTR LTR

SASDCMV

Poly A

gagpol

tatrev

RREψ

Vettori lentivirali

assenza dei fattori di virulenza vif, vpr, vpu, nef

rev in un plasmide separato: ridotta probabilità di ricombinazione

tat non necessaria se trascrizione attivata da CMV

Reverse transcription

gene 1

LTR

U3 R

LTR

U3 R U5

PBS PPT

U5

ψ

Trascrizione inversa

gene 1

LTR

U3 R

LTR

U3 R U5

Vettori SIN (self-inactivating)

PBS PPT

U5

ψ

Variazioni nella costruzione dei vettori gammaretrovirali

la delezione in U3 viene mantenuta quando il genoma viene retrotrascritto, il che distrugge l’attività di promotore/enhancer del LTR

Retroviruses integrate near transcriptionally active regions of DNA

Acceptor sites for retroviral integrations map near DNase I hypersensitive sites in chromatin (S. Vijaya et al. J. Virol. 1986)

Retrovirus integration and chromatin structure: Moloney murine leukemia proviral integration sites map near DNase I-hypersensitive sites (H. Rohdewohld et al. J. Virol. 1987)

Chromosome structure and human immunodeficiency type 1 cDNA integration: centromeric alphoid repeats are a disfavored target (S. Carteu et al. J. Virol. 1998)

HIV-1 integration in the human genome favors active genes and local hotspots (A.R.W. Schroder et al. Cell 2002)

MLV HIV-1

5 kb upstream 5 kb downstreamTranscribed gene

Integration is not random

MLV: transcriptional start site

HIV: transcriptional units

used Cre recombinase-mediated insertion of a codon-optimized HIV-1 gag-pol construct into a constitutivelyexpressed locus in 293FT cells. The remaining vectorcomponents were then transfected into a clone thatexpressed high levels of gag-pol. This approach solved akey issue with lentiviral vector production found in pre-vious studies, which was that plasmid transfection of thegag-pol cassette did not induce continuous high-levelexpression. The availability of stable packaging cell linescould potentially expedite the development and manu-facture of new lentiviral vector-based gene therapies as wellas reduce the costs of this critical component of genetherapy. However, toxicity induced from expression of theother viral proteins used for packaging (i.e., Rev or VSV-G)also remains a challenge.

Clinical use of lentiviral vectors

Lentiviral and retroviral vectors are important technologiesthat are currently in development for a number of clinicalapplications requiring transfer of genetic material (Fig. 4).Lentiviral vectors have become particularly attractive forclinical applications due to their ability to more efficientlytransduce non-proliferating or slowly proliferating cells,such as CD34+ stem cells. The first application of lentiviralvectors in the clinical setting used a conditionallyreplication-competent lentiviral vector encoding an anti-sense RNA targeting the HIV envelope gene. This vectorwas used to transduce mature peripheral blood T cells forthe treatment of natural HIV infection. No adverse eventsattributable to the lentiviral vector were reported in this trial,which included up to 8 years of follow-up in some patients[66]. In addition, integration site analysis demonstratedpreferential integration within transcribed genes as expec-ted, with no significant change in the distribution of inte-gration sites in the pre-infusion cellular product andengrafted T cells. Lentiviral vector-based gene transfer intoCD34+HSCs has subsequently been applied in the treat-ment of several genetic diseases, including β-thalassemia[67], X-linked adrenoleukodystrophy [51], metachromaticleukodystrophy [68, 69], and Wiskott-Aldrich Syndrome[70]. No adverse events related to the vector have beenreported in these trials. In the initial study of HSCs trans-duced with β-globin in patients with β-thalassemia, onepatient with βE/β0-thalassemia achieved independence fromtransfusion [67]. Interestingly, this response was associatedwith a relative increase in a dominant myeloid clone bearinga lentiviral vector insertion within the HMGA2 gene locus.It is unknown whether the insertion within this dominantclone was merely a coincidence or selected based onenhanced proliferation resulting from dysregulation of theHMGA2 gene. Several studies using lentiviral vectors to

modify HSCs continue (Table 1), and longer follow-up willbe necessary to fully establish the safety of lentiviral vectorsin this therapeutic setting.

The safety of lentiviral vectors in ex vivo gene transferinto HSCs remains a somewhat open question; however, thefield has now gained over 10 years of experience withlentiviral vectors for gene transfer into mature T cells. Therehave been several recent advances in cancer immunotherapyusing genetically modified T cells. One approach involvesthe generation of cytotoxic T cells through the transductionof a tumor-specific TCR into a patient’s own T cells. Cur-rently, ongoing phase 1 and phase 2 clinical trials are usingautologous T cells that have been transduced to express thetumor antigens NY-ESO-1, MART-1, WT-1, and others[71]. In a trial using a lentiviral vector to transfer a TCRspecific for a peptide that was shared by NY-ESO-1 andLAGE-1 as a therapy for patients with multiple myeloma,clinical responses were observed in 16 of 20 patients, withminimal safety concerns [72]. In addition, a lentiviral vectorexhibited better transduction efficiency than a gammare-troviral vector in transducing T cells with a TCR targetingthe Melan-A/MART-1 antigen [73], and a phase 2 trial ofT cells transduced with a lentivirus to express MART-1 inpatients with metastatic melanoma is ongoing. In sometrials using TCR-modified cells, adverse effects caused bythe transferred T cells have been observed; however, atpresent, there are no reports of adverse effects attributable tothe use of lentiviral vectors in these studies.

In a similar approach to using TCRs to reprogram T cells,CARs targeting the B-cell marker CD19 introduced into

Table 1 Ongoing clinical trials using lentiviral vectors to modifyhematopoietic stem cells

Condition Phase NCT number

Transfusion-dependent β-thalassemia 1/2 NCT02453477

3 NCT02906202

Cerebral adrenoleukodystrophy 2/3 NCT01896102

Sickle cell disease 1 NCT02140554

1 NCT02193191

Metachromatic leukodystrophy andadrenoleukodystrophy

1/2 NCT02559830

Wiskott-Aldrich syndrome 1/2 NCT01347346

1/2 NCT01347242

1/2 NCT02333760

X-SCID 1/2 NCT01306019

1/2 NCT01512888

ADA-SCID 1/2 NCT02999984

1/2 NCT01380990

Fanconi anemia 2 NCT02931071

X-linked chronic granulomatous disease 1/2 NCT02234934

ADA adenosine deaminase, SCID severe combined immunodeficiency

Clinical use of lentiviral vectors 1535

Leukemia (2018) 32:1529–1541https://doi.org/10.1038/s41375-018-0106-0

REVIEW ARTICLE

Acute lymphoblastic leukemia

Clinical use of lentiviral vectors

Michael C. Milone1,2 ● Una O’Doherty1

Received: 14 November 2017 / Revised: 21 February 2018 / Accepted: 2 March 2018 / Published online: 22 March 2018© The Author(s) 2018. This article is published with open access

AbstractViral vectors provide an efficient means for modification of eukaryotic cells, and their use is now commonplace in academiclaboratories and industry for both research and clinical gene therapy applications. Lentiviral vectors, derived from the humanimmunodeficiency virus, have been extensively investigated and optimized over the past two decades. Third-generation, self-inactivating lentiviral vectors have recently been used in multiple clinical trials to introduce genes into hematopoietic stem cellsto correct primary immunodeficiencies and hemoglobinopathies. These vectors have also been used to introduce genes intomature T cells to generate immunity to cancer through the delivery of chimeric antigen receptors (CARs) or cloned T-cellreceptors. CAR T-cell therapies engineered using lentiviral vectors have demonstrated noteworthy clinical success in patientswith B-cell malignancies leading to regulatory approval of the first genetically engineered cellular therapy using lentiviralvectors. In this review, we discuss several aspects of lentiviral vectors that will be of interest to clinicians, including an overviewof lentiviral vector development, the current uses of viral vectors as therapy for primary immunodeficiencies and cancers, large-scale manufacturing of lentiviral vectors, and long-term follow-up of patients treated with gene therapy products.

Introduction

The advent of molecular biology in the 1970s enabled thedevelopment of a variety of tools to manipulate nucleic acidsand has transformed modern medicine. Molecular biologyforms the foundation of numerous biotherapeutics, such asrecombinant enzymes (e.g., factor IX in hemophilia), mono-clonal antibodies (e.g., trastuzumab), and growth factors (e.g.,erythropoietin). Gene therapy, which involves the delivery ofDNA encoding a gene of interest into a cell with the intentionof treating a disease, extends the power of molecular biologyto potentially correct diseases such as those caused by geneticdeficiencies (e.g., β-thalassemia due to a defect in the β-globingene). Beyond correcting genetic deficiencies, gene therapycan also endow a cell or organism with capabilities not pre-sent in the natural state. Adoptive cellular therapy usinggenetically engineered T cells is one of the most notable

examples. Using a synthetic gene, such as a chimeric antigenreceptor (CAR) or cloned T-cell receptor (TCR), T cells canbe endowed with the ability to recognize antigens that are notnaturally recognized by their endogenous TCRs. Thisapproach is capable of generating robust clinical responseseven in patients with advanced B-cell malignancies that arehighly refractory to other existing therapies [1].

Gene therapy via gammaretroviruses, lentiviruses, adeno-viruses, and adeno-associated viruses is attractive because of thenatural ability of viruses to enter into and deliver geneticmaterial to cells [2]. Gammaretroviruses and lentiviruses aresubtypes of retroviruses, which contain an RNA genome that isconverted to DNA in the transduced cell by a virally encodedenzyme called reverse transcriptase. Although the use of gam-maretroviral vectors is more common, especially in the researchsetting, the number of clinical trials using lentiviral vectors forgene therapy is increasing. This review discusses the develop-ment of lentiviral vectors and summarizes their current clinicalinvestigation, particularly from a safety perspective.

History of lentiviral vector development

Lentivirus biology

The basic genes required for retroviral and lentiviral sur-vival and function are the gag, pol, and env genes; gag

* Michael C. Milonemilone@mail.med.upenn.edu

1 Department of Pathology and Laboratory Medicine, PerelmanSchool of Medicine at the University of Pennsylvania,Philadelphia, PA, USA

2 Center for Cellular Immunotherapies, Perelman School ofMedicine at the University of Pennsylvania, Philadelphia, PA,USA

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Third-generation / Self-Inactivating (SIN): In a third-generation vector, the 3’ LTR is modified, with tat being eliminated and rev provided in a separate plasmid. Since the HIV promoter in the 5’ LTR depends on tat, a vector without tat needs to have its wild-type promoter replaced with a heterologous enhancer/promoter to ensure transcription. Such promoter could be either viral (like CMV) or cellular (like EF1-α).

Lentiviral Vector Generations Summary Table

First Generation

Second Generation

Third Generation

Plasmids

3

3

4

Deletion in 3’ LTR - SIN

No

No

Yes

Packaging plasmids with HIV genes

1

1

2

Accessory genes: vif, vpr, vpu, nef

All absent

All absent

All absent

tat and rev genes

On a single packaging plasmid

On a single packaging plasmid

tat is absent; rev on a separate plasmid

gag and pol genes

Same plasmid

Same plasmid

Same plasmid

Recombination events needed to generate Replication Competent Lentiviruses (RCL)*

2 recombinations

3 recombinations

4 recombinations between plasmids

without homology & must pick a promoter

to complement SIN deletion

* The risk of formation of RCLs exists not only during lentiviral vector production, but also during experiments involving materials infected with wild-type HIV. Recombination between the lentiviral vector and HIV can lead to the generation of new viruses with unknown safety

Transfer vector

Transgene

Ψ

+

Packaging plasmids

gag, pol + env (het.)

Envelope plasmid

+ rev

Lentiviral Vector Generations Summary Table

Pro and cons of lentiviral vectors

Can carry large transgenes (up to 8 Kb)

Efficient gene transfer

Infects dividing and non-dividing cells

No immunogenic proteins generated

Stable integration into the host genome and stable expression of the transgene

Potential for generation of RCL

Potential for insertional mutagenesis: Even replication-incompetent lentiviruses with human tropism are able to infect human cells and integrate their genome into the host cellsàrisk in case of accidental exposure

In vivo inactivation by the complement system

Do not work in all tissues (muscle, heart, vessels)

Real applications for ex vivo gene therapy (HSC, epithelia)

No packaging cells for scaling up

www.thelancet.com/haematology Vol 6 May 2019 e239

Articles

Lentiviral haemopoietic stem/progenitor cell gene therapy for treatment of Wiskott-Aldrich syndrome: interim results of a non-randomised, open-label, phase 1/2 clinical studyFrancesca Ferrua*, Maria Pia Cicalese*, Stefania Galimberti, Stefania Giannelli, Francesca Dionisio, Federica Barzaghi, Maddalena Migliavacca, Maria Ester Bernardo, Valeria Calbi, Andrea Angelo Assanelli, Marcella Facchini, Claudia Fossati, Elena Albertazzi, Samantha Scaramuzza, Immacolata Brigida, Serena Scala, Luca Basso-Ricci, Roberta Pajno, Miriam Casiraghi, Daniele Canarutto, Federica Andrea Salerio, Michael H Albert, Antonella Bartoli, Hermann M Wolf, Rossana Fiori, Paolo Silvani, Salvatore Gattillo, Anna Villa, Luca Biasco, Christopher Dott, Emily J Culme-Seymour, Koenraad van Rossem, Gillian Atkinson, Maria Grazia Valsecchi, Maria Grazia Roncarolo, Fabio Ciceri, Luigi Naldini, Alessandro Aiuti

SummaryBackground Wiskott-Aldrich syndrome is a rare, life-threatening, X-linked primary immunodeficiency characterised by microthrombocytopenia, infections, eczema, autoimmunity, and malignant disease. Lentiviral vector-mediated haemopoietic stem/progenitor cell (HSPC) gene therapy is a potentially curative treatment that represents an alternative to allogeneic HSPC transplantation. Here, we report safety and efficacy data from an interim analysis of patients with severe Wiskott-Aldrich syndrome who received lentiviral vector-derived gene therapy.

Methods We did a non-randomised, open-label, phase 1/2 clinical study in paediatric patients with severe Wiskott-Aldrich syndrome, defined by either WAS gene mutation or absent Wiskott-Aldrich syndrome protein (WASP)expression or a Zhu clinical score of 3 or higher. We included patients who had no HLA-identical sibling donor available or, for children younger than 5 years of age, no suitable 10/10 matched unrelated donor or 6/6 unrelated cord blood donor. After treatment with rituximab and a reduced-intensity conditioning regimen of busulfan and fludarabine, patients received one intravenous infusion of autologous CD34+ cells genetically modified with a lentiviral vector encoding for human WAS cDNA. The primary safety endpoints were safety of the conditioning regimen and safety of lentiviral gene transfer into HSPCs. The primary efficacy endpoints were overall survival, sustained engraftment of genetically corrected HSPCs, expression of vector-derived WASP, improved T-cell function, antigen-specific responses to vaccinations, and improved platelet count and mean platelet volume normalisation. This interim analysis was done when the first six patients treated had completed at least 3 years of follow-up. The planned analyses are presented for the intention-to-treat population. This trial is registered with ClinicalTrials.gov (number NCT01515462) and EudraCT (number 2009-017346-32).

Findings Between April 20, 2010, and Feb 26, 2015, nine patients (all male) were enrolled of whom one was excluded after screening; the age range of the eight treated children was 1·1–12·4 years. At the time of the interim analysis (data cutoff April 29, 2016), median follow-up was 3∙6 years (range 0∙5–5∙6). Overall survival was 100%. Engraftment of genetically corrected HSPCs was successful and sustained in all patients. The fraction of WASP-positive lymphocytes increased from a median of 3∙9% (range 1·8–35·6) before gene therapy to 66∙7% (55·7–98·6) at 12 months after gene therapy, whereas WASP-positive platelets increased from 19∙1% (range 4·1–31·0) to 76∙6% (53·1–98·4). Improvement of immune function was shown by normalisation of in-vitro T-cell function and successful discontinuation of immunoglobulin supplementation in seven patients with follow-up longer than 1 year, followed by positive antigen-specific response to vaccination. Severe infections fell from 2∙38 (95% CI 1·44–3·72) per patient-year of observation (PYO) in the year before gene therapy to 0∙31 (0·04–1·11) per PYO in the second year after gene therapy and 0∙17 (0·00–0·93) per PYO in the third year after gene therapy. Before gene therapy, platelet counts were lower than 20 × 10⁹ per L in seven of eight patients. At the last follow-up visit, the platelet count had increased to 20–50 × 10⁹ per L in one patient, 50–100 × 10⁹ per L in five patients, and more than 100 × 10⁹ per L in two patients, which resulted in independence from platelet transfusions and absence of severe bleeding events. 27 serious adverse events in six patients occurred after gene therapy, 23 (85%) of which were infectious (pyrexia [five events in three patients], device-related infections, including one case of sepsis [ four events in three patients], and gastroenteritis, including one case due to rotavirus [three events in two patients]); these occurred mainly in the first 6 months of follow-up. No adverse reactions to the investigational drug product and no abnormal clonal proliferation or leukaemia were reported after gene therapy.

Interpretation Data from this study show that gene therapy provides a valuable treatment option for patients with severe Wiskott-Aldrich syndrome, particularly for those who do not have a suitable HSPC donor available.

Funding Italian Telethon Foundation, GlaxoSmithKline, and Orchard Therapeutics.

Lancet Haematol 2019; 6: e239–53

Published Online April 10, 2019 http://dx.doi.org/10.1016/S2352-3026(19)30021-3

See Comment page e230

*Contributed equally

San Raffaele Telethon Institute for Gene Therapy (SR-Tiget) (F Ferrua MD, M P Cicalese MD, S Giannelli PhD, F Dionisio MSc, F Barzaghi MD, M Migliavacca MD, M E Bernardo MD, V Calbi MD, M Facchini PhD, C Fossati MSc, E Albertazzi PhD, S Scaramuzza PhD, I Brigida PhD, S Scala PhD, L Basso-Ricci MSc, M Casiraghi RN, F A Salerio MSc, A Villa MD, L Biasco PhD, Prof L Naldini MD, Prof A Aiuti MD), Pediatric Immunohematology and Bone Marrow Transplantation Unit (F Ferrua, M P Cicalese, F Barzaghi, M Migliavacca, M E Bernardo, V Calbi, R Pajno MD, M Casiraghi, D Canarutto MD, Prof A Aiuti), Hematology and Bone Marrow Transplantation Unit (A A Assanelli MD, Prof F Ciceri MD), Department of Anesthesia and Critical Care (R Fiori MD, P Silvani MD), and Blood Transfusion Service (S Gattillo MD), IRCCS San Raffaele Scientific Institute, Milan, Italy; Vita-Salute San Raffaele University, Milan, Italy (F Ferrua, R Pajno, D Canarutto, Prof F Ciceri, Prof L Naldini, Prof A Aiuti); Center of Biostatistics for Clinical Epidemiology, University of Milano—Bicocca, Monza, Italy (S Galimberti PhD, Prof M G Valsecchi PhD); Department of Pediatric Hematology/Oncology, Dr von Haunersches University Children’s Hospital, Munich,

www.thelancet.com/haematology Vol 6 May 2019 e239

Articles

Lentiviral haemopoietic stem/progenitor cell gene therapy for treatment of Wiskott-Aldrich syndrome: interim results of a non-randomised, open-label, phase 1/2 clinical studyFrancesca Ferrua*, Maria Pia Cicalese*, Stefania Galimberti, Stefania Giannelli, Francesca Dionisio, Federica Barzaghi, Maddalena Migliavacca, Maria Ester Bernardo, Valeria Calbi, Andrea Angelo Assanelli, Marcella Facchini, Claudia Fossati, Elena Albertazzi, Samantha Scaramuzza, Immacolata Brigida, Serena Scala, Luca Basso-Ricci, Roberta Pajno, Miriam Casiraghi, Daniele Canarutto, Federica Andrea Salerio, Michael H Albert, Antonella Bartoli, Hermann M Wolf, Rossana Fiori, Paolo Silvani, Salvatore Gattillo, Anna Villa, Luca Biasco, Christopher Dott, Emily J Culme-Seymour, Koenraad van Rossem, Gillian Atkinson, Maria Grazia Valsecchi, Maria Grazia Roncarolo, Fabio Ciceri, Luigi Naldini, Alessandro Aiuti

SummaryBackground Wiskott-Aldrich syndrome is a rare, life-threatening, X-linked primary immunodeficiency characterised by microthrombocytopenia, infections, eczema, autoimmunity, and malignant disease. Lentiviral vector-mediated haemopoietic stem/progenitor cell (HSPC) gene therapy is a potentially curative treatment that represents an alternative to allogeneic HSPC transplantation. Here, we report safety and efficacy data from an interim analysis of patients with severe Wiskott-Aldrich syndrome who received lentiviral vector-derived gene therapy.

Methods We did a non-randomised, open-label, phase 1/2 clinical study in paediatric patients with severe Wiskott-Aldrich syndrome, defined by either WAS gene mutation or absent Wiskott-Aldrich syndrome protein (WASP)expression or a Zhu clinical score of 3 or higher. We included patients who had no HLA-identical sibling donor available or, for children younger than 5 years of age, no suitable 10/10 matched unrelated donor or 6/6 unrelated cord blood donor. After treatment with rituximab and a reduced-intensity conditioning regimen of busulfan and fludarabine, patients received one intravenous infusion of autologous CD34+ cells genetically modified with a lentiviral vector encoding for human WAS cDNA. The primary safety endpoints were safety of the conditioning regimen and safety of lentiviral gene transfer into HSPCs. The primary efficacy endpoints were overall survival, sustained engraftment of genetically corrected HSPCs, expression of vector-derived WASP, improved T-cell function, antigen-specific responses to vaccinations, and improved platelet count and mean platelet volume normalisation. This interim analysis was done when the first six patients treated had completed at least 3 years of follow-up. The planned analyses are presented for the intention-to-treat population. This trial is registered with ClinicalTrials.gov (number NCT01515462) and EudraCT (number 2009-017346-32).

Findings Between April 20, 2010, and Feb 26, 2015, nine patients (all male) were enrolled of whom one was excluded after screening; the age range of the eight treated children was 1·1–12·4 years. At the time of the interim analysis (data cutoff April 29, 2016), median follow-up was 3∙6 years (range 0∙5–5∙6). Overall survival was 100%. Engraftment of genetically corrected HSPCs was successful and sustained in all patients. The fraction of WASP-positive lymphocytes increased from a median of 3∙9% (range 1·8–35·6) before gene therapy to 66∙7% (55·7–98·6) at 12 months after gene therapy, whereas WASP-positive platelets increased from 19∙1% (range 4·1–31·0) to 76∙6% (53·1–98·4). Improvement of immune function was shown by normalisation of in-vitro T-cell function and successful discontinuation of immunoglobulin supplementation in seven patients with follow-up longer than 1 year, followed by positive antigen-specific response to vaccination. Severe infections fell from 2∙38 (95% CI 1·44–3·72) per patient-year of observation (PYO) in the year before gene therapy to 0∙31 (0·04–1·11) per PYO in the second year after gene therapy and 0∙17 (0·00–0·93) per PYO in the third year after gene therapy. Before gene therapy, platelet counts were lower than 20 × 10⁹ per L in seven of eight patients. At the last follow-up visit, the platelet count had increased to 20–50 × 10⁹ per L in one patient, 50–100 × 10⁹ per L in five patients, and more than 100 × 10⁹ per L in two patients, which resulted in independence from platelet transfusions and absence of severe bleeding events. 27 serious adverse events in six patients occurred after gene therapy, 23 (85%) of which were infectious (pyrexia [five events in three patients], device-related infections, including one case of sepsis [ four events in three patients], and gastroenteritis, including one case due to rotavirus [three events in two patients]); these occurred mainly in the first 6 months of follow-up. No adverse reactions to the investigational drug product and no abnormal clonal proliferation or leukaemia were reported after gene therapy.

Interpretation Data from this study show that gene therapy provides a valuable treatment option for patients with severe Wiskott-Aldrich syndrome, particularly for those who do not have a suitable HSPC donor available.

Funding Italian Telethon Foundation, GlaxoSmithKline, and Orchard Therapeutics.

Lancet Haematol 2019; 6: e239–53

Published Online April 10, 2019 http://dx.doi.org/10.1016/S2352-3026(19)30021-3

See Comment page e230

*Contributed equally

San Raffaele Telethon Institute for Gene Therapy (SR-Tiget) (F Ferrua MD, M P Cicalese MD, S Giannelli PhD, F Dionisio MSc, F Barzaghi MD, M Migliavacca MD, M E Bernardo MD, V Calbi MD, M Facchini PhD, C Fossati MSc, E Albertazzi PhD, S Scaramuzza PhD, I Brigida PhD, S Scala PhD, L Basso-Ricci MSc, M Casiraghi RN, F A Salerio MSc, A Villa MD, L Biasco PhD, Prof L Naldini MD, Prof A Aiuti MD), Pediatric Immunohematology and Bone Marrow Transplantation Unit (F Ferrua, M P Cicalese, F Barzaghi, M Migliavacca, M E Bernardo, V Calbi, R Pajno MD, M Casiraghi, D Canarutto MD, Prof A Aiuti), Hematology and Bone Marrow Transplantation Unit (A A Assanelli MD, Prof F Ciceri MD), Department of Anesthesia and Critical Care (R Fiori MD, P Silvani MD), and Blood Transfusion Service (S Gattillo MD), IRCCS San Raffaele Scientific Institute, Milan, Italy; Vita-Salute San Raffaele University, Milan, Italy (F Ferrua, R Pajno, D Canarutto, Prof F Ciceri, Prof L Naldini, Prof A Aiuti); Center of Biostatistics for Clinical Epidemiology, University of Milano—Bicocca, Monza, Italy (S Galimberti PhD, Prof M G Valsecchi PhD); Department of Pediatric Hematology/Oncology, Dr von Haunersches University Children’s Hospital, Munich,