RESEARCH ARTICLE SUMMARY

Lentiviral Hematopoietic Stem Cell READ THE FULL ARTICLE ONLINE

http://dx.doi.org/10.1126/science.1233158

Gene Therapy Benefits Metachromatic Cite this article as A. Biffi et al.,

Science 341, 1233158 (2013).
DOI: 10.1126/science.1233158

Leukodystrophy
Alessandra Biffi,* Eugenio Montini, Laura Lorioli, Martina Cesani, Francesca Fumagalli,
Tiziana Plati, Cristina Baldoli, Sabata Martino, Andrea Calabria, Sabrina Canale, Fabrizio FIGURES IN THE FULL ARTICLE
Benedicenti, Giuliana Vallanti, Luca Biasco, Simone Leo, Nabil Kabbara, Gianluigi Zanetti,
Fig. 1. Gene marking in patients after HSC-GT.
William B. Rizzo, Nalini A. L. Mehta, Maria Pia Cicalese, Miriam Casiraghi, Jaap J. Boelens,
Ubaldo Del Carro, David J. Dow, Manfred Schmidt, Andrea Assanelli, Victor Neduva, Clelia Di Fig. 2. ARSA expression in patients after
Serio, Elia Stupka, Jason Gardner, Christof von Kalle, Claudio Bordignon, Fabio Ciceri, Attilio HSC-GT.
Rovelli, Maria Grazia Roncarolo, Alessandro Aiuti, Maria Sessa, Luigi Naldini* Fig. 3. Clinical follow up of MLD patients after
HSC-GT.
Introduction: Metachromatic leukodystrophy (MLD) is a neurodegenerative lysosomal storage dis- Fig. 4. LV genomic integration profile.

Downloaded from www.sciencemag.org on July 2, 2015

ease caused by arylsulfatase A (ARSA) deficiency. The disease primarily affects children and invari- Fig. 5. Common insertion site analysis.
ably leads to premature death. In previous work with a mouse model of MLD, we used a lentiviral
vector (LV) to introduce a functional ARSA gene into hematopoietic stem cells (HSCs) ex vivo and Fig. 6. Stem cell marking and clonal dynamics.
showed that reinfusion of the engineered HSCs prevented and corrected disease manifestations
in the animals. To determine whether this gene therapy strategy is safe and can offer therapeutic SUPPLEMENTARY MATERIALS
benefit to patients with early-onset MLD, we designed a phase I/II trial. www.sciencemag.org/cgi/content/full/
Methods: We optimized LV manufacturing and HSC transduction in clinical grade conditions. Three science.1233158/DC1
children with ARSA deficiency and mutations associated with early-onset MLD were treated at the Materials and Methods
presymptomatic stage; all had at least one older sibling affected by the same disease variant. HSCs Figs. S1 to S22
from the patients were transduced ex vivo with a LV carrying the ARSA gene. Tables S1 to S19
Patients were treated with a myeloablative busulfan conditioning regimen before reinfusion of References
the engineered HSCs. Clinical and objective evaluations were collected up to 24 months after treat-
ment. Molecular follow-up of vector integration site distribution was performed on hematopoietic RELATED ITEMS IN SCIENCE
cells derived from bone marrow and peripheral blood. Aiuti et al., Lentiviral hematopoietic stem cell
Results: There was high-level stable engraftment of the transduced HSCs in the bone marrow and gene therapy in patients with Wiskott-Aldrich
peripheral blood of all patients at all times tested, with 45 to 80% of bone marrow–derived hema- syndrome. Science 341, 1233151 (2013).
topoietic colonies harboring the vector. With these high gene marking levels, ARSA activity was doi:10.1126/science.1233151
reconstituted to above normal values in the hematopoietic lineages and in the cerebrospinal fluid. I. Verma, Edging toward the promise of gene
Analysis of >36,000 different LV integration sites showed that the high gene marking was sus- therapy. Science 341, 853–855 (2013).
tained by highly polyclonal engraftment of transduced cells without evidence of aberrant clonal doi:10.1126/science.1242551
behavior. Several integration sites were shared among progenitors and mature myeloid, B- and
T-cells sampled at long intervals after treatment, indicat-
ing efficient transduction and engraftment of HSCs. These
findings were associated with a clear therapeutic benefit
because the disease did not progress in any of the treated
patients, even after the time of onset projected from sib-
ling cases.
Discussion: Our gene therapy protocol allows stable
engraftment of transduced HSCs at high levels and with-
out evidence of vector-induced genotoxicity. The reconsti-
tution of ARSA activity in the cerebrospinal fluid and the
arrested progression of neurodegenerative disease in the
three treated patients demonstrate that the transplanted
cells, or their progeny, can seed the nervous system and
deliver therapeutic levels of active enzyme. Although our
data are promising, long-term follow-up of the patients is
needed in order to establish the full therapeutic potential HSC gene therapy can prevent progression of metachromatic leukodystrophy. Magnetic reso-
nance (MR) images of the brain of a patient (MLD01) before and after gene therapy. The brain of this
of this gene therapy strategy for MLD. In addition, our data
patient appeared largely normal 2 years after treatment. In contrast, the brain of an untreated, age-
position LV gene transfer as a feasible means to engineer matched late infantile MLD patient (UT LI MLD) showed severe demyelination associated with diffuse
human hematopoiesis to its near entirety—an approach atrophy. (Top) Axial T2 weighted fast spin-echo MR images. (Bottom) Fluid-attenuated inversion recovery
that could be exploited for treatment of other diseases. (FLAIR) MR images.

The list of author affiliations is available in the full article online.

*Corresponding author. E-mail: biffi[email protected] (A.B.); [email protected] (L.N.)

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Published by AAAS
 RESEARCH ARTICLE
risk of genotoxic insertions because of their ad-
vanced design and the integration site selection
typical of the parental HIV (19, 20), it remained
Lentiviral Hematopoietic Stem possible that increasing the total integration load
in the infused HSPCs would offset the biosafety
Cell Gene Therapy Benefits advantage.
In the present study, we optimized LV-mediated

Metachromatic Leukodystrophy gene transfer into human HSCs for clinical trans-
lation and used this HSC-GT protocol to treat
nine patients with early onset MLD in a phase I/II
Alessandra Biffi,1,2,3*† Eugenio Montini,1* Laura Lorioli,1,2,3,4 Martina Cesani,1 trial. Here, we report the outcome of the treat-
Francesca Fumagalli,2,4,5 Tiziana Plati,1 Cristina Baldoli,6 Sabata Martino,7 Andrea Calabria,1 ment in the first three treated patients at 24 months
Sabrina Canale,2 Fabrizio Benedicenti,1 Giuliana Vallanti,8 Luca Biasco,1 Simone Leo,9 follow-up for patient MLD01 and 18 months
Nabil Kabbara,10 Gianluigi Zanetti,9 William B. Rizzo,11 Nalini A. L. Mehta,12 follow-up for patients MLD02 and MLD03.
Maria Pia Cicalese,2,3 Miriam Casiraghi,2 Jaap J. Boelens,13 Ubaldo Del Carro,5 David J. Dow,12
Manfred Schmidt,14 Andrea Assanelli,3,15 Victor Neduva,12 Clelia Di Serio,4 Elia Stupka,16 Results
Jason Gardner,17 Christof von Kalle,14 Claudio Bordignon,4,8 Fabio Ciceri,3,15 Attilio Rovelli,18
Maria Grazia Roncarolo,1,2,3,4 Alessandro Aiuti,1,2,3,19 Maria Sessa,2,5 Luigi Naldini1,4† Efficient ex Vivo Transfer of the ARSA Gene
into the HSPCs of MLD Patients
Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disease caused by Transduction of human bone marrow (BM)–
arylsulfatase A (ARSA) deficiency. Patients with MLD exhibit progressive motor and cognitive derived CD34+ cells was optimized to reach ≥2
impairment and die within a few years of symptom onset. We used a lentiviral vector to transfer vector copy number per genome (VCN) (fig. S1),
a functional ARSA gene into hematopoietic stem cells (HSCs) from three presymptomatic patients based on the ARSA overexpression levels re-
who showed genetic, biochemical, and neurophysiological evidence of late infantile MLD. quired for therapeutic efficacy in preclinical studies
After reinfusion of the gene-corrected HSCs, the patients showed extensive and stable ARSA (8, 9, 21, 22). CD34+ cells were stimulated ex vivo
gene replacement, which led to high enzyme expression throughout hematopoietic lineages and with early acting cytokines in serum-free medi-
in cerebrospinal fluid. Analyses of vector integrations revealed no evidence of aberrant clonal um and transduced with purified third-generation
behavior. The disease did not manifest or progress in the three patients 7 to 21 months beyond LVs encoding the human ARSA cDNA under the
the predicted age of symptom onset. These findings indicate that extensive genetic engineering control of the human phosphoglycerate kinase
of human hematopoiesis can be achieved with lentiviral vectors and that this approach may promoter (PGK) (fig. S2). VCN was measured in
offer therapeutic benefit for MLD patients. the progeny of the treated cells obtained by means
of bulk liquid culture, colony-forming assays and
etachromatic leukodystrophy (MLD) Achieving a high level of functional gene replace- repopulation of chimeric Rag2−/− gchain−/− mice.

M is an autosomal recessive lysosomal

storage disease caused by mutations in
the ARSA gene that result in a deficiency of the
ment is particularly important in the setting of
HSC-GT; to date, gene replacement in the hem-
atopoietic cells of treated patients has been lim-
1
San Raffaele Telethon Institute for Gene Therapy (TIGET), San
Raffaele Scientific Institute, 20132 Milan, Italy. 2TIGET Pediatric
enzyme arylsulfatase A (1). ARSA deficiency ited (12–15). Clinical Research Unit, Division of Regenerative Medicine, Stem
causes accumulation of the enzyme substrate In a mouse model of MLD, we have dem- Cells and Gene Therapy, San Raffaele Scientific Institute, 20132
Milan, Italy. 3Pediatric Immunohematology and Bone Marrow
sulfatide in oligodendrocytes, microglia, and cer- onstrated that disease manifestations can be Transplant Unit, San Raffaele Scientific Institute, 20132 Milan,
tain neurons of the central nervous system (CNS), prevented and corrected with lentiviral vector Italy. 4Vita-Salute San Raffaele University, 20132 Milan, Italy.
5
and in Schwann cells and macrophages of the (LV)–based HSC-GT but not HSCT (8, 9, 16). Neurology Unit, Department of Neurology, San Raffaele Sci-
peripheral nervous system (PNS). This build-up This is consistent with the observation that entific Institute, 20132 Milan, Italy. 6Neuroradiology Unit, Head
of sulfatide leads to widespread demyelination HSCT fails to provide consistent benefits in MLD and Neck Department, San Raffaele Scientific Institute, 20132
Milan, Italy. 7Department of Experimental Medicine and Bio-
and neurodegeneration, which is ultimately mani- patients (3, 5–7). LV-based HSC-GT induced ex- chemical Sciences, University of Perugia, 06122 Perugia, Italy.
fested in patients as severe progressive motor and tensive and supraphysiological expression of the 8
MolMed, 20132 Milan, Italy. 9Distributed Computing Group,
cognitive impairment. MLD is classified into clin- functional ARSA gene throughout the HSC proge- Center for Advanced Studies, Research and Development in
ical variants according to the age of symptom ny, which in turn mediated widespread cross- Sardinia (CRS4), 09010 Pula, Italy. 10Pediatric Hematology On-
cology Division, Rafic Hariri University Hospital, Beirut, Lebanon.
onset. Patients with the late-infantile (LI) variant correction of CNS and PNS resident cells (8, 9). 11
Department of Pediatrics, University of Nebraska Medical
show symptoms within the second year of life, Translating this strategy into a clinical pro- Center, Omaha, NE 68198, USA. 12Molecular and Cellular Tech-
have the most severe manifestations of the dis- tocol, however, posed several challenges, includ- nologies, GlaxoSmithKline, Stevenage 5G1 2NY, UK. 13Pediatric
ease, and die within a few years of symptom on- ing gene transfer efficacy and safety. Although Blood and Marrow Transplantation Program, University Medical
Center 3584 CX Utrecht, Netherlands. 14Department of Transla-
set. Current treatments do not substantially delay stable marking (10 to 15%) was reported in two tional Oncology, National Center for Tumor Diseases and German
the progression or fatal outcome of MLD (2, 3). patients with X-linked adrenoleukodystrophy Cancer Research Center, 69120 Heidelberg, Germany. 15He-
Hematopoietic stem cell transplantation (HSCT) (X-ALD) in the first trial of LV-mediated HSC- matology and Bone Marrow Transplant Unit, San Raffaele
and HSC gene therapy (HSC-GT) have been in- GT (17), it was not clear whether LVs could trans- Scientific Institute, 20132 Milan, Italy. 16Center for Transla-
tional Genomics and BioInformatics, San Raffaele Scientific
vestigated for the treatment of lysosomal storage duce human HSCs in clinically relevant settings Institute, 20132 Milan, Italy. 17Regenerative Medicine Discov-
diseases because enzyme-proficient hematopoietic with the efficiency required for application to MLD ery Performance Unit, GlaxoSmithKline Research and Devel-
progeny cells can migrate to the affected tissues, gene therapy. Vector safety is also a concern in opment, King of Prussia, PA 19406, USA. 18Bone Marrow
including the CNS, scavenge the stored material, light of the clinical experience with g-retroviral Transplant Unit, MBBM Foundation, Pediatric Department,
such as sulfatide in MLD, and cross-correct the vectors, which showed that genome-wide vector Milano-Bicocca University at San Gerardo Hospital, 20052
Monza, Italy. 19University of Rome Tor Vergata, 00133 Rome,
resident cells (4). The efficacy of these treatments integration in hematopoietic stem/progenitor cells Italy.
varies among different types of lysosomal storage (HSPCs) has the potential to trigger leukemia *These authors contributed equally to this work.
diseases (3, 5–7) and with the extent of functional through insertional mutagenesis (14, 18). Although †Corresponding author. E-mail: [email protected] (A.B.);
gene expression in the transplanted cells (8–11). several studies have shown that LVs reduce the [email protected] (L.N.)

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RESEARCH ARTICLE
A summary of the LV manufacturing process matological parameters matching the normal val- matches the data measured on the in vitro prog-
(flow chart, main steps, and yields) as well as the ues for age. No abnormal expansion or clonal eny of the infused cells. Correspondingly high and
specifications set for testing the good manufac- outgrowth was detected in the peripheral blood stable gene marking was observed in the circulat-
turing practice (GMP) lots released for this study (PB) and BM by cell type composition, cytologic, ing CD15+ granulocytes and CD14+ monocytes
are reported in fig. S3 and tables S1 and S2. karyotypic, or immunophenotypic studies (fig. (Fig. 1C), and in myeloid cells and progenitors
After optimizing the gene transfer procedures S5). A mild (maximum National Cancer Institute/ isolated from the BM (fig. S6), beginning with
for human cells, we designed a clinical trial of National Institutes of Health common toxicity cri- the first sampling and throughout the follow-up.
HSC-GT in which autologous HSPCs from pa- teria grade 2) transient increase in hepatocellular A progressive increase in gene marking of lym-
tients with MLD would be transduced ex vivo enzyme levels was detected within 2 to 3 weeks phoid cells was observed—more rapid for B and
with ARSA-encoding LVs and then reinfused after after treatment. Serious adverse events included NK cells and delayed for the longer-lived T cells—
the patients had been treated by a myeloablative two cases of central venous catheter–related in- as expected from the choice of the conditioning
regimen using the alkylating agent busulfan (fig. fection, which promptly resolved upon antibio- regimen (Fig. 1C and fig. S6). The different re-
S4). Because MLD patients usually express resid- tic treatment. Replication competent LVs (RCLs), population kinetics of hematopoietic lineages by
ual, nonfunctional ARSA protein, they are unlike- antibodies to HIV Gag p24, and antibodies to the transduced HSCs accounts for the progressive
ly to mount an immune response toward ARSA. ARSA in patients’ PB were negative at all times increase of the gene-marking level observed in
Our trial design therefore did not include immuno- tested. total PB mononuclear cells (PBMCs) (Fig. 1D).
suppressive agents. The high level of gene marking drove recon-
Three presymptomatic MLD patients, bio- High-Level Engraftment of the Transduced stitution of the defective ARSA activity up to
chemically characterized for ARSA deficiency HSPCs and Sustained ARSA Reconstitution above-normal values in the most therapeutically
and carrying mutations associated with LI MLD, Beginning 1 month after transplant, we observed relevant myeloid populations (Fig. 2, A and B) as
were enrolled and treated; all patients had one or high-level stable engraftment of the transduced well as in other circulating cells (fig. S7, A and
more older siblings with LI MLD onset within 2 cells in the BM and PB of all patients at all times B). ARSA protein was isolated from patient hem-
years of age (table S3). At enrollment, patients tested (Fig. 1A). Between 45 and 80% of the atopoietic cells as early as 1 month after treatment
were defined as presymptomatic based on the lack colonies outgrown from patients’ BM in the colony- (fig. S7C) at above-normal expression levels in
of obvious clinical signs of the disease, indepen- forming cell (CFC) assay harbored the LV ge- all tested samples (fig. S7D). The enzyme iso-
dent of the presence of abnormalities in instru- nome (Fig. 1A). Quantitative polymerase chain lated from patient cells was found to hydrolyze
mental tests [electroneurographic recordings (ENG) reaction (PCR) on CD34+ cells sorted from the the natural substrate sulfatide in vitro, confirming
and/or brain magnetic resonance imaging (MRI)]. patients’ BM showed stable gene marking with full functionality (0.003 mU of ARSA isolated from
Treatment of the patients began 2 to 12 months VCN ranging from 0.9 to 1.9 up to the latest follow- normal donor and treated patients’ hematopoietic
before the reported age of onset of the disease in up time (Fig. 1B). Considering the fraction of cells hydrolyzed 3.6 T 1.2 nmol of substrate). In
their affected matched siblings (23–25). cells harboring the vector (estimated from the situ hybridization for LV mRNA and immuno-
Twenty to 30 days after collection of an HSC percentage of clonogenic progenitors outgrown fluorescence for ARSA expression costained a high
back-up, a BM harvest was performed followed from the patients’ BM positive for LV DNA), proportion of patients’ blood cells consistently with
by isolation of CD34+ cells by means of standard these values imply a VCN of 2 to 4 in the en- the high VCN detected by means of quantitative
immunomagnetic procedures. Transduction of pa- grafted transduced CD34+ cells, which closely PCR in these cells (fig. S8 and table S6). Func-
tients’ CD34+ cells was performed as described
above. Cells were washed, resuspended, and kept
at +4°C until successful completion of a first
panel of fast quality control (QC) tests (table S4).
The transduced CD34+ cells were then released
and infused fresh intravenously. Other relevant
QC results (table S4) became available 2 to 5 weeks
after infusion. VCN ranged from 2.5 to 4.4, trans-
duction efficiency was 90 to 97%, and ARSA ac-
tivity was reconstituted at ≥10-fold the level
measured in healthy controls in the cultured prog-
eny of the infused cells (fig. S1 and table S5).
A myeloablative, dose-adjusted busulfan reg-
imen [target area under the curve (AUC) 4800
mg/L*hour] (table S5) was administered intravenous-
ly to the patients from day –4 up to day –1 before
HSC-GT in a total of 14 doses, with the last busulfan
dose administered ≥24 hours before trans-
duced cell infusion. The conditioning regimen
was well tolerated. The patients experienced severe
neutropenia (absolute neutrophil count < 500/ml)
from as early as day +9 up to day +45 after trans-
plant at the latest (fig. S5 and table S5). No re-
duction or only a minor transient reduction of
lymphocyte counts was observed in the absence
of immunosuppressive/lymphotoxic drugs in the Fig. 1. Gene marking in patients after HSC-GT. (A) Engraftment of the transduced cells, evaluated
pretransplant conditioning (fig. S5). Patients re- with quantitative PCR on individual colonies from CFC assay performed on PB- and BM-derived cells and
quired transfusional support up to day +45 after expressed as percentage (%) of LV+ colonies on total tested colonies. (B to D) VCN expressed as copies of
treatment for thrombocytopenia and anemia (fig. LV/human genome measured with quantitative PCR on BM-derived CD34+ cells (B), individual subpop-
S5). Thereafter, the patients rapidly recovered he- ulations isolated from PB of patient MLD01 (C), and total PBMCs from patients MLD01, -02 and -03 (D).

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 RESEARCH ARTICLE
tional ARSAwas isolated from cerebrospinal fluid by 30 months of age, when they were wheelchair- age of 39 months has been reported to display
(CSF) collected from all three patients 1 year (and bound and unable to support their head and trunk clinical features as positive as those we observed
2 years for MLD01) after HSC-GT at levels and (table S7). In contrast, at 39 months of age MLD01 in MLD01.
activity comparable with those obtained from healthy was able to stand independently and to walk and Patients MLD02 and MLD03 remain fully
donor samples, whereas no ARSA protein could run with single aid (either the hand of another asymptomatic, with normal motor and cognitive
be isolated from CSF before HSC-GT treatment individual or an external walking device) (level 2 development for their ages (30 and 25 months,
(Fig. 2, C and D). These data demonstrate stable of the GMF-C MLD scale) (table S7). His Gross respectively); however, the clinical follow-up pe-
above-normal ARSA expression throughout the hem- Motor Function Measure (GMFM) score pro- riod for these patients after the predicted time of
atopoietic lineages and efficient delivery and bio- gressively increased from enrollment (GMFM symptom onset was shorter than that for MLD01.
availability of the enzyme in the CNS after HSC-GT. score of 164) up to a score of 193 at 39 months, At the latest clinical follow-up (18 months after
which was substantially higher than all the scores treatment), MLD02 was 7 months beyond and
HSC-GT Provides Therapeutic Benefit to recorded in our age-matched historical LI-MLD MLD03 was 10 months beyond the predicted
MLD Patients cohort [5.5 on average (26)], suggesting a con- time of symptom onset. Both patients had a
Clinical observation and objective evaluations tinuous motor development (Fig. 3A and table GMF-C MLD score of 0 at the last evaluation,
were collected up to 24 months after treatment S8). Cognitive evaluation by the Bayley Scale of which is in sharp contrast with the disease evo-
for MLD01 and up to 18 months after treatment Infant and Toddler Development revealed a nor- lution documented in their affected siblings at the
for MLD02 and MLD03. At the last follow-up, mal composite IQ score for chronological age, corresponding age (table S7). Their GMFM scores
MLD01, MLD02, and MLD03 were 39, 30, and with normal language and cognitive abilities (table progressively increased after treatment, which is
25 months old, respectively (table S3). Because S9) at all tested time points, including the last consistent with the acquisition of normal devel-
disease onset in the LI variant occurs invariably one at 39 months of age, an age at which the two opmental motor milestones for age (Fig. 3A and
before 24 months of age and clinical manifes- affected siblings were incapable of any voluntary table S8). Furthermore, in both patients the Bayley
tations are highly homogeneous, in particular among speech. The preexisting severe peripheral neuro- Scale of Infant and Toddler Development showed
siblings (23, 24), all three patients in our study pathy of MLD01 [nerve conduction velocity (NCV) a normal composite IQ score for the age at all
were evaluated beyond their expected age of dis- index (26) –11.5 at baseline] improved after treat- times tested (table S9). The ENG findings for both
ease onset. ment (NCV index –7.1 at last follow-up) (Fig. 3B). patients remained stable from baseline up to the
MLD01 is particularly informative because the Brain MRI showed slight signal inhomogeneity, latest follow-up; MLD02 showed near normal
follow-up time for this patient is longest and be- present since baseline evaluation and stable there- motor and sensory conduction velocities since
cause the predicted time of symptom onset was after, and a small area of hyper-intensity in the baseline, whereas MLD03 showed a stable mild
earlier than that of the other two patients. This corpus callosum that appeared at the 12-month demyelinating neuropathy (Fig. 3B). Brain MRI
patient’s two older siblings manifested regression follow-up and remained stable thereafter. All revealed that both patients had a normal progres-
of psychomotor performance and arrest of acqui- other brain MR findings were normal for age (Fig. sion of white matter signal due to myelination up
sition of new motor and cognitive skills at 18 months 3C). In contrast, untreated LI-MLD patients invar- to the latest follow-up. MLD02 had small areas of
of age. Consistent with LI-MLD natural history iably develop extensive and severe demyelination T2 and fluid-attenuated inversion recovery (FLAIR)
(26), both siblings had rapid disease progression associated with diffuse atrophy by 24 to 36 months hyper-intensity signal in posterior and anterior peri-
after onset and reached level 6 of the Gross Mo- of age (Fig. 3C) (26, 28). To our knowledge, no ventricular white matter, present since baseline and
tor Function Scale for MLD (GMF-C MLD) (27) unequivocally diagnosed LI-MLD patient at the stable up to the latest follow-up (fig. S9).

Fig. 2. ARSA expression

in patients after HSC-GT.
(A and B) ARSA activity mea-
sured with the p-nitrocatechol
sulfate (PNC) assay on CD15+
cells (A) and CD14+ cells (B)
isolated from the patients’
PB. The activity range mea-
sured in a cohort of healthy
donors (HDs) (n ≥ 10 subjects)
is shown. (C) Representative
DEAEcellulose-chromatography
analysis on 500 ml of cerebro-
spinal fluid (CSF) from a pool
of four HDs, of a represent-
ative MLD patient before
treatment (MLD01 pre-GT)
andofthesamepatient1year
after gene therapy. The peak
of activity corresponds to
the native form of the ARSA
enzyme, as also demonstrated
by its absence/very low re-
sidual activity in the patient’s
pretreatment sample. (D) Spe-
cific activity (toward MUS) of
the ARSA enzyme isolated from the CSF of HDs (two cohorts of four donors each) and of the treated MLD patients (circles, MLD01; blue diamond, MLD 02; and
green triangle, MLD03) sampled 12 months (red circle, MLD01; MLD02 and -03 were also sampled at 12 months) and 24 months (pink circle, MLD01) after
treatment.

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Polyclonal Reconstitution of Hematopoiesis PB cells, and subsets (table S11) at 1, 3, 6, 9, and MLD02 (1,518,802), and MLD03 (425,356)
Without Evidence of Vector Genotoxicity 12 months for all patients and, for MLD01 and on the human genome (NCBI HG19). Because
To determine whether there was any cause for MLD02, 18 months after HSC-GT (table S12). many identical ISs were retrieved from multiple
concern regarding the safety of the LV in these Vector-genome junctions were retrieved by means lineages and/or time points, the number of distinct
patients, we monitored the clonal dynamics of of linear amplification mediated–PCR (LAM-PCR) ISs was 14,482, 11,077, and 10,959 for patients
hematopoietic repopulation at the molecular level by using three different restriction enzymes (fig. MLD01, MLD02 and MLD03, respectively. The
by analyzing the vector genomic integrations in S12). From the three patients, >800 independent genomic distribution of IS, both in the in vitro–
the patients’ reconstituted hematopoietic cells (figs. LAM-PCRs were generated and sequenced by cultured CD34+ cells and in vivo, matched the
S10 and S11, table S10, and materials and meth- 454-Roche (Basel, Switzerland) and Illumina (San previously reported LV preference for integration
ods). Because of the genome-wide quasi–random Diego, California) technologies to obtain >10 × 106 within transcriptional units (on average, 80% of
integration profile of LV, each insertion creates a sequence reads. Using a bioinformatics analysis IS were within genes) (fig. S14). To identify the
different genetic marker that can be used to track pipeline and collision/contamination filtering (fig. gene classes preferentially targeted by LV inte-
the clonal behavior of individual transduced cells. S10, fig. S13, and table S13, A to C), we mapped grations, we analyzed IS data sets from either the
Genomic DNA was obtained from whole BM, integration sites (IS) for patient MLD01 (1,120,414), in vitro progeny of the infused CD34+ cells or a
pool of all in vivo–harvested samples for each
patient using the GREAT (Genomic Regions En-
richment of Annotations Tool) software (table S14,
A to F). In all patients, the gene classes of chro-
matin modification/remodeling, major histocom-
patibility complex class II–related functions, steroid
hormone receptors, and RNA processing were
overrepresented (table S14, A to F). These gene
classes were the same, or shared >75% genes,
with gene classes overrepresented in the ALD
HSC-GT (17, 29) (Fig. 4A and fig. S15).
We measured the proportion of sequencing
reads representing each IS within our data sets as
a surrogate readout for the relative abundance of
the cell clone harboring that integration at a given
time. For each time point, we analyzed the IS data
sets from the CD34+, myeloid (pooled CD13+,
CD14+, and CD15+ data sets), B (CD19+ ) and
T cells (CD3+). Almost every cell clone marked
by a specific IS accounted for only a fraction of a
percent of the total clones at any given time. A
few IS-marked cell clones showed a higher per-
centage at one time point but then disappeared or
were strongly reduced at later time points (Fig. 4,
B and C, and fig. S16). From these results, we
conclude that no clonal dominance events occurred
in these patients.
To determine whether other hallmarks of in-
sertional mutagenesis were present in the patients,
we assessed the occurrence of common insertion
sites (CISs), insertional hotspots that may result
from integration bias at the time of transduction
or in vivo selection of clones harboring integra-
tions that confer growth advantage. CISs were
Fig. 3. Clinical follow up of MLD patients after HSC-GT. (A and B) GMFM score (A) and NCV index (B) identified with an algorithm based on Abel et al.
of the three treated patients and of a historical cohort of LI-MLD patients (gray circles). The dotted lines (30) and by the Grubbs test for outliers (29). Both
indicate for each treated patient (inset, color code) the expected time of disease onset, according to the analyses showed that the MLD CISs were mostly
disease onset observed in their affected matched siblings; n.r., normal range of the NCV index. (C) Axial distributed in clusters within gene-dense regions
T2 weighted fast spin-echo MR images (top) and FLAIR MR images (bottom) obtained from patient MLD01 heavily targeted by LV integrations (Fig. 5A, figs.
at baseline (before GT) and at +2 years after treatment, and corresponding (equivalent) images of an age-
S17 to S19, and tables S15 and S16), many of
matched untreated patient with LI-MLD (in parenthesis, the chronological age at imaging acquisition in
which had already been described in the ALD
months). In MLD01 images, a small area of hyperintensity is present within the splenium of the corpus
callosum, stable in extension and appearance as compared with that in the +12 months follow-up; subtle HSC-GT trial (Fig. 5B) (17, 29). Given the larger
signal inhomogeneities are present within posterior periventricular and posterior centrum semiovale size of the MLD data set, it contained almost the
white matter, in the absence of focal lesions; these inhomogeneities, present since baseline as just barely entire ALD CIS data set. Moreover, the Grubbs
T2 and FLAIR hyperintensity signal, are (now) more evident because of the normal signal of the test for outliers when applied to the genomic re-
surrounding myelin; subarachnoid and ventricular spaces are within normal limits, even if a little wider gions surrounding each CIS confirmed that the
when compared with that of the baseline. Basal ganglia and thalami remain of normal appearance. In UT CIS from the MLD clinical trial were not signif-
LI-MLD images, extensive, diffuse symmetric hyperintensities with typical “tigroid pattern” are seen within icantly over-targeted with respect to the neighbor-
periventricular white matter, centrum semiovale, corpus callosum, external and internal capsules, and ing genes, with OPTC being the only exception
cerebellar deep white matter. A severe diffuse brain atrophy involving basal ganglia and thalamy, which (fig. S19F and table S16). Cell clones with IS
show T2 hypointense signal, is also present. targeting CIS genes did not represent the most

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abundant IS-marked cell clones at any given time sets representing progenitors and mature myeloid creased with follow-up time (Fig. 6B). We then
point of the follow-up. IS targeting OPTC were and mature lymphoid cells in each patient. After plotted the occurrence of shared ISs in each sub-
not detected in samples from the longest follow- stringent filtering to reduce the false discovery set of cells over time, considering the B and T
up time point. rate due to the impurity of each cell fraction ana- cells separately, and ranked them for multiple hits
lyzed and the occurrence of collisions during IS (Fig. 6C and table S18). The increasing presence
Extensive HSC Gene Marking in Vivo processing (fig. S20 and table S17), a fraction of over time of CD34+ progenitors and mature cells
To assess HSC gene marking in vivo, we ana- ISs were consistently shared among the three data of myeloid and lymphoid lineages marked by
lyzed the subset of IS shared among three data sets of each patient (Fig. 6A). This fraction in- identical integrations is evidence of self-renewal

Fig. 4. LV genomic inte-

gration profile. (A) Gene on-
tology (GO) analysis on ISs
from three patients with MLD
and two patients with ALD who
had been treated with HSC-GT
(17). This analysis was performed
using the GREAT software (http://
bejerano.stanford.edu/great/).
The genomic positions of ISs
in each data set were weighted
by the binomial test in order
to evaluate the clustering at
given genomic regions and
by the hypergeometric test in
order to evaluate the overrep-
resentation of gene functions.
GO classes were considered sig-
nificant when both tests pro-
vided a false discovery rate of
<0.05. To compare the level
of similarity between the gene
classes preferentially targeted
by LV integrations in ALD and
MLD clinical trials, we counted
the number of shared genes
contained in the significantly
overrepresented gene classes
for GO molecular functions of
each clinical trial (results for
GO biological processes are
shown in fig. S13). The extent
of gene sharing between the
overrepresented gene classes
are indicated by a color scale.
(B) Box plot of the percent-
age of sequence reads (y axis)
for unequivocally mapped ISs
from patients MLD01 and MLD02
in different cell types (CD34+,
myeloid, or lymphoid cells)
from different sources (BM or
PB) and time points (months
after gene therapy, indicated
below the x axis). The num-
ber of reads for each IS was
normalized to the total num-
ber of sequence reads from the
same time point and source.
ISs over the 95 percentile of
the data set are shown as dots distinct from the box and whiskers, which are same lineage so as to emphasize the maximum relative contribution, thus
mostly flattened to the bottom of the plot. The total number of ISs for each obtaining values that are greater or equal to percentages shown in (B). For each
lineage and time point is shown on top. Most represented integrations (with IS, colored cells indicate retrieval at ≥5%, with higher color intensity indicating
the hit gene indicated next to the dot) are enriched in oligoclonal populations, higher percentage, whereas gray cells indicate retrieval at low percentage
such as in PB-derived B or T cells at early time points. (C) Follow-up of the most (from 0.006% to <5%). Lack of color indicates that the integration was not
represented ISs during time in different cell types. Percentages of sequence retrieved at the indicated time point and source. The targeted genes are in-
reads for each IS are calculated on the total number of sequence reads from the dicated. Almost all integrations reached ≥5% at only one time point.

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and multilineage potential of the transduced en- creased and stabilized. PB myeloid cells showed findings ascertained through brain imaging and
grafted HSCs. high and stable diversity, which is consistent with electrophysiological and biochemical studies and
To estimate the number of HSCs contributing that of the progenitor pool, whereas B and T cell is in contrast with the disease evolution observed
to hematopoietic reconstitution, we used the frac- diversity increased with time and stabilized 6 in (i) the patients’ older affected siblings with
tion of ISs shared between independent samplings months after gene therapy. Patient MLD03 showed matched ARSA mutations and related genetic
of the same cell population (supplementary lower IS sharing between lineages and decreas- background and (ii) our historical cohort of un-
materials, materials and methods). For this analy- ing diversity of myeloid cells as compared with treated LI-MLD patients. MRI analysis detected
sis, we considered the output of short-lived mature the other two patients, possibly because of the a minor demyelinating lesion in the brain of MLD01
myeloid cells in the PB long-term after HSC-GT shorter follow-up and the higher VCN of the 1 year after treatment. This minor lesion, which
as readout of total HSC activity at that time. We infused cells. might have arisen before the infused cells reached
compared ISs shared between CD14+ and CD15+ Overall, these data provide evidence that ef- a sufficient CNS engraftment to exert beneficial
PB cells at months 9, 12, and 18 after HSC-GT ficient ex vivo gene transfer was followed by sub- effects, remained stable at all subsequent follow-
by the mark-recapture approach using the Petersen/ stantial engraftment and sustained clonogenic ups. This finding is clearly different from what is
Schnabel estimator method (31) and Chao Poisson activity of the transduced HSCs in the patients, observed in untreated LI-MLD patients, who show
regression model (table S19). The resulting lower resulting in extensive polyclonal reconstitution rapidly progressive and widespread brain demye-
bound population size estimates were 3.7 × 103 of hematopoiesis with gene-corrected cells. lination. The severe peripheral neuropathy already
to 5.7 × 103 in patient MLD01 and 2.1 × 103 to present in MLD01 at the time of HSC-GT improved
3.4 × 103 in MLD02, indicating an abundant pool Discussion after treatment, with amelioration of the amplitude
of engrafted transduced self-renewing progeni- LI MLD is a devastating disease that invariably of motor action potentials and of the nerve con-
tors in both patients. leads to death of affected children within a few duction velocities after treatment. These findings
To quantify clonal diversity in each lineage years of symptom onset. We have shown that HSC- were associated with clinical evidence of normal
over time, we calculated the Shannon Diversity GT halted disease manifestation and/or progression cognitive evolution and continuous improvements
Index (Fig. 6D and fig. S22). This index mea- in three presymptomatic children with LI-MLD in motor skills. Although the follow-up is shorter
sures the entropy of an IS data set taking into for follow-up times ranging from 18 to 24 months for MLD02 and MLD03, these patients remained
account the total number of ISs and their relative after treatment (7 to 21 months after predicted asymptomatic at a chronological age when their
contribution. The diversity of CD34+ progenitors disease onset). disease was expected to have manifested.
was lower early after HSC-GT as compared with The clinical benefit observed in the three pa- In previous work with the MLD mouse mod-
that of the infused cell population and then in- tients was accompanied by measurable objective el, we showed that the therapeutic benefit of

Fig. 5. Common insertion site analysis. (A) Frequency distribution along chromosomes (top track) of RefSeq genes (second track below),
LV integrations for each patient (third, fourth, and fifth track below for MLD01, MLD02, and MLD03, respectively). The last three tracks at
the bottom represent Manhattan plots of the P values of the gene integration frequencies evaluated with the genome-wide Grubbs test for
outliers. The dashed lines indicate the upper limit required to reach CIS significance. CISs are enriched in gene dense regions targeted at
high frequency by means of LV integrations. (B) Venn diagrams showing the overlap between CIS genes in the MLD and ALD HSC-GT trials.

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HSC-GT arises from the homing of ARSA- of microglia with donor-derived cells was shown stringent lot-release criteria so as to ensure efficient
overexpressing HSPCs and/or their myeloid prog- after myeloablation and intravenous infusion of transduction while limiting detrimental effects on
eny to the CNS and PNS, where they contribute HSPCs (8, 33, 34). The latter findings are con- HSPCs. Second, we validated the transduction
to the replacement of a substantial fraction of sistent with clinical evidence that HSCT can pro- protocol for reproducible high-rate gene transfer
microglia/resident macrophages, scavenge the stored vide therapeutic benefit in certain lysosomal into BM-derived mouse-repopulating human
sulfatide, and establish a local source of func- storage diseases affecting the CNS, and with the HSPCs and infused fresh cells after transduction
tional enzyme bioavailable throughout the neural few available studies investigating the donor to avoid incurring in any cell loss associated with
tissues. Although we could not directly probe our origin of microglia cells within the brains of trans- freeze/thawing. Last, we administered a dose-
patients for the migration of gene-modified cells planted humans (35, 36). We speculate that adjusted myeloablative regimen before HSPC
into the CNS, we documented that CSF samples high-level engraftment of the myeloid compart- infusion to foster engraftment.
from the patients contained substantial levels of ment in PB and BM by the transduced HSPCs is Another key goal of our study was to assess
ARSA protein and activity long after HSC-GT. likely needed to support their efficient migration the long-term safety of HSC gene transfer. Using
This objective finding, together with the halted to the neural tissues. It is possible, however, that deep-sequencing and bioinformatics, we found
progression of neural disease, suggests that the CNS myeloid progenitors, rather than mature mono- no evidence that cell clones harboring ISs under-
and PNS were seeded by gene-corrected myeloid cytes, are primarily responsible for seeding the go in vivo expansion or selection. Rather, our data
cells upon HSC-GT. CNS in our patients (34). suggest that the genomic distribution of IS in pa-
Recent studies in mice have shown that mi- At least three factors may have contributed to tients’ hematopoietic cells reflects the LV inte-
croglia mostly originates from an early seeding of the high levels of gene replacement observed in gration in the infused HSPCs. These findings are
neural tissues by embryonic lineage macrophages patients’ hematopoiesis. First, we optimized the in sharp contrast with those reported at compa-
followed by in situ turnover under homeostatic LV-manufacturing process, aiming to produce vec- rable times after HSC-GT in some other trials
conditions (32). However, substantial replacement tor with high titer, infectivity, and purity using using g-RV; in those trials, cell clones harboring

Fig. 6. Stem cell marking and clonal dynamics. Analysis of IS data was row represents a specific IS, with colored bars indicating retrieval from the indicated
performed after filtering for collision and sample impurity. (A) Venn diagrams cell lineage and time point after gene therapy (columns). The line color varies with
showing the sharing of ISs between CD34+ cells and lymphoid and myeloid the degree of sharing among lineages (red, high level of sharing; blue, low level of
lineages. (B) Percentage of shared ISs (y axis) between BM-derived CD34+ and sharing; white, no integration retrieved). PB, peripheral blood; B, B cells; T, T cells;
myeloid or lymphoid lineages during time (months after GT, indicated below). (C) Myelo, myeloid cells. (D) Shannon Diversity Index (y axis) was calculated to mea-
Tracking of ISs shared between multiple lineages with time in patient MLD01. Each sure the clonal diversity of hematopoietic reconstitution during time.

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ISs that target oncogenes underwent expansion ClinicalTrials.gov #NCT01560182. The parents a self-inactivating lentiviral vector produced with
or selection in vivo and eventually progressed to of all subjects provided written informed con- a third-generation split packaging system and
leukemia (14, 18, 37–40). The LV IS distribution sent for experimental treatment. Biological sam- pseudotyped by the vesicular stomatitis virus G
in MLD patients overlaps with that reported for ples were obtained from MLD patients—healthy protein (VSV.G) (6). The vector lots were pro-
ALD patients after LV-based HSC-GT, a reassur- children and adults as controls—with approval duced by MolMed (Milan, Italy) using a large-scale
ing finding considering that no adverse effect of of the San Raffaele Scientific Institute’s Ethics validated process. The vectors were produced
gene transfer has been reported in that trial after Committee and consent from parents or subjects. following Good Manufacturing Practice (GMP)
more than 5 years of follow-up. A similar inte- guidelines, except for initial studies of gene trans-
gration profile in the absence of clonal expansion Neurological and Motor Evaluation fer protocol optimization, which used pre-GMP
was observed in three patients with Wiskott- A standard neurological evaluation was performed (GMP-like/large scale produced) vector. The pro-
Aldrich Syndrome treated by LV-based HSC-GT in all patients for assessing the presence of disease tocol for vector production is detailed in fig. S3.
with a follow-up of up to 2.5 years (47). Overall, clinical signs. In addition, we applied GMFM Briefly, the ARSA LV was produced by means of
these data strongly support the improved safety (26, 42) and the Gross Motor Function Classi- transient four-plasmid transfection of 293T cells.
of LV comprising self-inactivating long terminal fication for MLD (GMFC-MLD) (27) for prop- 293T cells were derived from human embryonic
repeat and moderately active internal promoters, er quantification of patients’ motor abilities. The kidney 293 cells with stable transfection of the
which makes it unlikely that an insertion would ac- same trained personnel applied the scales at each temperature-sensitive SV40 T-antigen; a subclone
tivate a nearby oncogene (19, 20, 41). Nevertheless, evaluation. of the 293T (from Stanford University) selected
we will continue longer-term evaluation of the at the Salk Institute (La Jolla, California) for its high-
treated and additional patients for full assessment Neuropsychological Evaluation yield performance in production of lentiviral vec-
of the risk-benefit ratio and therapeutic potential Neuropsychological evaluation was performed by tors through transient transfection was used for
of HSC-GT. using the Bayley Scale for Infant and Toddler MCB establishment; a 293T MCB was generated
The stability of gene marking together with Development (BSID-II) (43), applied at baseline at Genethon (Paris, France) in GMP conditions.
the identification of several ISs shared among evaluation and then every 6 months. 293T cells from the master cell bank were ex-
progenitors, mature myeloid cells, and both ma- panded in T162 flasks and then in 10-tray cell
jor lymphoid lineages long-term after HSC-GT Neurophysiological Evaluation factories and transiently transfected by means of
provide evidence that bona fide HSCs were trans- Sensory conduction of sural and median nerves, calcium phosphate precipitation with four plas-
duced. Furthermore, the estimated large number and motor conduction of deep peroneal and ulnar mids encoding for two core packaging constructs
of active HSCs would imply that a good fraction of nerves were studied and a z score for NCV was (pKLGag/pol and pKRev), the envelope con-
harvested HSCs were transduced and long-term calculated for each examined nerve (patient’s struct (pK.G), and the transfer vector construct
engrafted the patients. These data position LV gene NCV – normal donors’ mean NCV/normal do- (pARSA). Twenty-four hours after removal of
transfer as a feasible means to engineer human nors’ NCV standard deviation); an NCV index the transfection medium, the cell supernatant was
hematopoiesis to its near entirety, an approach that was calculated as the average of z scores obtained harvested and stored at 4°C. The culture medium
may be exploited to develop novel treatments from the four tested nerves for each patient per was replaced, and after a further 24 hours, a sec-
for several inherited and acquired diseases. time, as described (26). ond harvest was performed. The medium col-
lected from the two harvests was pooled and
Materials and Methods MR Examination filtered through 5/0.45-mm filters so as to discard
All children underwent anatomical MRI on a 1.5 cell debris. The downstream purification process
Clinical Protocol and Patients Tesla scanner using a six-channel SENSE head included a benzonase treatment overnight at 4°C,
The Istituto Superiore di Sanità and the Ethical coil (Gyroscan Intera, Philips, Netherlands). MRI followed by a diethylamino anion exchange (DEAE)
Committee of the San Raffaele Scientific Institute scans were performed with Spin Echo T1 weighted chromatography step, concentration, and gel filtra-
in Milan approved the study. The study promoter images, repetition time (TR) 600 ms, echo time tion in CellGro medium. The benzonase treatment
is TIGET, San Raffaele Hospital, Italy and the (TE) 15 ms, rec matrix 288, 22 slices, 5 and 3 mm was aimed at degrading plasmid DNA and host
financial sponsor of the study is the Telethon thick, axial and sagittal planes; Turbo Spin Echo cell–derived DNA present in the clarified cell su-
Foundation. The medicinal product received T2-weighted images, TE120 ms, TR 5500 ms pernatant, thus facilitating their removal through
Orphan Drug Designation (ODD) (EMEA/OD/ 24 slices, axial, sagittal and coronal planes, subsequent purification steps. Anion exchange chro-
102/06) by the European Medicines Agency (EMA) 4.5 and 3 mm thick; fluid-attenuated inversion matography involved the absorption of negative-
for the treatment of MLD. recovery Turbo Spin Echo, TE 140 ms, 24 slices, ly charged lentiviral particles to positively charged
We enrolled and treated three presymptomatic 4.5 mm thick; and volumetric T1 sequence (fast chromatographic support; viral particles were eluted
LI-MLD patients, who were classified as LI ac- field echo, 110 slices, 1.4 mm thick, and 0 mm by increasing ionic strength. This step was aimed
cording to the disease in their affected older sib- gap). MR examinations were performed under at the removal of contaminants such as host cell
lings using the historical classification based on sedation by a senior experienced pediatric an- proteins and serum-derived proteins such as bo-
age at symptom onset (table S3). The patients were esthesiologist (44); sedation was induced with vine serum albumin (BSA). Gel filtration, also
from Lebanon, the United States, and Egypt. Upon intravenous 1 mg/kg propofol and maintained defined as size exclusion chromatography, relies
obtaining informed consent, their diagnosis was with 4 mg/kg/hour propofol continuous infu- on the inability of the large lentiviral particles to
molecularly and biochemically confirmed. In all sion. Throughout MRI examination, saturation of be retained by matrix pores. This step was aimed
patients, the definitive MLD diagnosis was based peripheral oxygen (SpO2) was monitored with a at the removal of salts and small-sized contam-
on pathologic low ARSA activity and the pres- MRI-compatible device (Magnitude, Invivo Cor- inants such as DNA fragments as well as at me-
ence of disease-causing ARSA gene mutations on poration, Orlando, Florida). Desaturation (SpO2 < dium exchange. The resulting LV preparation, in
both alleles. Presymptomatic condition at enroll- 95%) was recorded together with any adverse CellGro medium, underwent one sterilizing
ment was defined on the basis of a normal neuro- event requiring the intervention of the attending 0.2-mm filtration and aseptic filling. The purified
logical and cognitive examination, independently anesthesiologist. vector preparation was stored at –80°C. The
from the presence of abnormalities at neurophys- produced vector lots were characterized as de-
iological tests or brain imaging. Large-Scale Lentiviral Vector Production scribed in table S2. Physical titer was measured
Details on clinical study enrollment crite- The vector used in this study (pCCLsin.cPPT. by means of enzyme-linked immunosorbent assay
ria, endpoints, and study plan can be found at hPGK.hARSA.WPREmut6 - PGK.ARSA.LV) is (Perkin Elmer, Applied Biosystem, Foster City,

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California) for the detection of HIV-1 gag p24 mononuclear cells by means of positive selection cells/ml or 1.25 × 104 to 1 × 106 MNCs/ml
capsid protein. Infectious titer was measured with immunomagnetic beads according to the purified from BM or PB. After 14 days of culture,
through transduction of a human T cell line with manufacturer’s procedure (Miltenyi Biotec, colonies were counted, distinguishing myeloid
serial dilutions of vector and calculation of the Bergisch-Gladbach, Germany) in GMP conditions. and erythroid origin, and plucked for molecular
copies of integrated vector per cell by quantita- The purified cells were cultured in retronectin-coated analysis.
tive PCR. VueLife bags (American Fluoroseal, Gaithersburg,
Maryland) in serum-free medium supplemented Quantitative PCR
HSC Isolation and Transduction with GMP-grade cytokines and exposed twice to Genomic DNA was extracted from CD34+ liq-
For in Vitro Experiments GMP-grade–purified vector at multiplicity of infec- uid culture, peripheral blood mononuclear cells
Healthy donor (HD) bone marrow CD34+ cells tion of 100, for a total culture time of 60 hours. At (PBMCs), MNCs, PB, and BM subpopulations
were purchased from Lonza (Basel, Switzerland) the end of the transduction procedure, the trans- of MLD patient samples with QIAamp DNA
or obtained after written informed consent ac- duced CD34+ cells were harvested, washed with Blood Micro and Mini Kits (Qiagen, Hilden, Ger-
cording to standard ethical procedure and with Cell Grow medium, and resuspended in saline many). Single colonies plucked from the CFC as-
approval of the San Raffaele Institute Ethical Com- solution at the concentration of 2 × 106 to 10 × say were digested for 4 hours at 37°C in Lysis
mittee (protocol TIGET-01). CD34+ cells from 106/ml for patient’s infusion. At the end of trans- Buffer (Lauryl Ether 0.1%, TRIS/hydrochloride
three MLD patients not enrolled in the gene ther- duction, a fraction of the cells was collected for (HCL) 10 mM, 100 mg/ml Proteinase K). After
apy trial—diagnosed on the basis of clinical symp- clonogenic assays, flow cytometry, and in vitro proteinase K inactivation (10 min at 95°C), lysates
toms, ARSA activity, and genetic analysis—were culture, as above. Cells were kept at 4°C up to were centrifuged at 13,000 revolutions per min at
similarly obtained after written informed consent the time of infusion, upon batch release according 4°C for 15 min.
(protocol LDM1) at the time of diagnostic or ther- to quality control tests [results are available at LV sequences were detected by means of
apeutic procedures. CD34+ cells were purified infusion for viability, immunophenotype, en- quantitative PCR on 25 to 50 ng of total genomic
from mononuclear cells by means of positive se- dotoxin, large T Ag DNA, and mycoplasma DNA or 10 ml of lysate supernatants by using
lection with immunomagnetic beads according to (table S4)]. previously described primers and probe (22).
the manufacturer’s procedure (Miltenyi Biotec, Absolute quantifications were plotted on stan-
Bergisch-Gladbach, Germany). Soon after puri- Patients’ Conditioning dard curves prepared with serial dilutions of ge-
fication or thawing, CD34+ cells were placed in Patients received a total of 14 body weight–based nomic DNA or cell lysate from a T cell clone
culture on retronectin-coated non–tissue culture– doses of intravenous busulfan, given every 6 hours containing 1 copy HIV/cell.
treated wells (T100A Takara) in CellGro SCGM from day –4 to day –1. The busulfan plasmatic Engraftment of the transduced cells was eval-
medium (2001 CellGenix) at a concentration of levels were monitored with serial sampling fol- uated by means of quantitative PCR on individ-
1 × 106 cells/ml in the presence of cytokines lowing first and fifth infusion. The doses after the ual colonies from CFC assay performed on either
[interleukin-3 (IL-3, 60 ng/ml), thrombopoietin 5th and 10th were adjusted, if needed, accord- PB- or BM-derived cells and expressed as per-
(TPO, 100 ng/ml), stem cell factor (SCF, 300 ng/ml), ing to plasmatic Busulfan levels with an ideal centage (%) of LV+ (VCN ≥ 0.5) colonies on total
and Flt3 ligand (Flt3-L, 300 ng/ml); PeproTech, AUC of range 4200-5600 mg/L*hour, target tested colonies (≥ 250 cells/colony).
Rocky Hill, New Jersey] for 24 hours of prestimu- 4800 mg/L*hour. The interval between the end of
lation. Cells were then transduced with ARSA LV the last intravenous busulfan dose and final product Analysis of Vbeta Repertoire
[at a multiplicity of infection (MOI) of 100] for 14 infusion was ≥24 hours in order to allow safe in- The analysis of Vbeta repertoire of the T cell
hours. In the protocol that foresees two rounds of fusion of the stem cells. At day 0, after premed- receptor was performed on 50 ml of patients’ total
transduction, selected for clinical application, cells ication with clorfeniramine (0.25 mg/kg, max dose PB according to the manufacturing procedure
were washed for 10 hours in CellGro SCGM me- 10 mg) or equivalent drug, the resuspended CD34+ described in the V Beta kit; Beckman Coulter
dium additioned with cytokines and underwent a cell product was infused intravenously through (823497) (Fullerton, California). 10000 events were
second hit of transduction in the same conditions the central catheter. acquired with FACS Canto II (BD-Biosciences,
as the first. At the end of transduction, cells were During hospitalization, patients were cared Europe) and then analyzed with FlowJo (TreeStar,
counted and collected for clonogenic assays, flow for in an isolation unit and received supportive Ashland, Oregon). The percentage of each Vbeta pop-
cytometry, and in vivo studies. Remaining cells therapy according to local standards. ulation was compared with the range of expres-
were plated in Iscove’s modified Dulbecco’s me- sion obtained from the analysis of a pool of 85
dium (IMDM) –10% fetal bovine serum (FBS) Isolation of Hematopoietic Subpopulations healthy donors provided by the manufacturer.
with cytokines (IL-3, 60 ng/ml; IL-6, 60 ng/ml; from Patients’ Peripheral Blood
SCF, 300 ng/ml) and cultured for a total of 14 days. and Bone Marrow Immunophenotype Analysis
Thereafter, cells were collected for molecular and Starting from mononuclear cells (MNCs) ob- Analysis of the immunophenotype of patients’
biochemical studies. tained from Lymphoprep gradient, human bone total PB and BM was performed by Laboraf
marrow and peripheral blood subpopulations (Ospedale San Raffaele) as described by inter-
For Patients’ Treatment were obtained through positive selection of nal SOPs.
Please refer to fig. S2. Total bone marrow was the following markers (Miltenyi Biotec micro-
collected from the iliac crests under sterile con- beads): CD34, CD13, CD15, CD56, CD19, CD3, ARSA Activity Determination
ditions and using general anesthesia, on day –4, glycophorin A (GLYA), and CD61 from the p-Nitrocatechol Sulfate (PNCS)
according to an internal standard operating pro- BM, and CD15, CD14, CD56, CD19, and CD3 CD34+ liquid culture, PBMCs, MNCs, PB, and
cedure. The total collected volume was estimated from PB, according to manufacturer’s procedures. BM subpopulations of MLD patients and healthy
according to the content of CD34+ cells evaluated Purity of each subpopulation was checked by donors were resuspended in sodium acetate tri-
by previous bone marrow aspiration; on average, means of flow-cytometry. Data are reported in hydrate 0.05 M (500,000 cells in 25 ml). Samples
20 to 25 ml/kg patient’s body weight was col- table S11. were then sonicated and centrifuged, and the
lected. The harvested bone marrow—collected in supernatant was collected for ARSA activity and
a dedicated bag, sealed, and identified—was then Clonogenic Assay (CFC) protein quantification (Bradford assay; Biorad
transferred to MolMed S.p.A and kept in sterile The CFC assay was performed by means of plating #500-0006) (Hercules, California). ARSA ac-
conditions in a refrigerator at 4°C for 24 hours. in methylcellulose medium (human MethoCult; tivity was detected with PNCS as substrate, as
CD34+ cells were then purified on day –3 from StemCell Technologies) either 800 to 1000 CD34+ described, loading a maximum concentration of

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protein extract of 0.3 mg/ml diluted in sodium and then fixed with 4% paraformaldehyde (PFA) bodies were added: APC-conjugated anti-CD45,
acetate trihydrate 0.05 M (9). solution. PE-conjugated anti-CD34, anti-CD13, anti-CD4,
An antisense 598–base pair digoxigenin- anti-CD11b and TC-conjugated anti-CD19, anti-
DEAE-Cellulose Chromatography and labeled oligonucleotide complementary to W-Pre CD8, anti-CD3 under control of relative IgG iso-
4-Methyl-Umbellipheryl-Sulfate mRNA sequence and a control sense sequence type control (DAKO; BD-Biosciences).
Cells were harvested, washed in phosphate- were produced .The in situ protocol was modified After 20 min on ice, cells were washed,
buffered saline, and resuspended in 10 mM TRIS/ from (46). Cells were treated with prehybridiza- resuspended in PBS 2% FBS, 1% PFA for cy-
HCl buffer, pH 7.5, containing 0.1% (v/v) Nonidet tion protocol and incubated overnight at 37°C with tometric analysis. Of 100,000 events, 3000 were
NP40 detergent (Sigma-Aldrich, St. Louis, Missouri). 0.5 mg/ml probe. The probe was detected by sheep acquired with FACS Canto II (BD-Biosciences,
Cell lysates were subjected to three rounds of anti Digoxigenin 1:500 (Roche Applied Science, Europe) and then analyzed by FlowJo (TreeStar,
sonication. Procedures were carried out at 4°C. Penzberg, Germany) overnight 4°C. Ashland, Oregon). During quadrant analysis, only
Proteins were measured with the Bradford method Immunofluorescence on ARSA-hemagglutinin fluorescence that excluded >99.9% of isotype con-
by using bovine serum albumin as standard. (HA) (9)–transduced Hela cells and on patients’ trol events was considered to be specific.
Similar cell number (500,000 cells) was loaded and healthy donors’ PB cells or HeLa cells was
into a 0.2-ml DEAE chromatography column performed overnight at 4°C by using rabbit anti– Vector Integration Site Analysis
equilibrated with a 25 mM TRIS/HCl buffer, pH Human Lamp1 1:100 (Abcam), goat anti–Human A detailed description of the materials and meth-
7.5. The flow rate was 0.2 ml/min. After washing ARSA 1:50 (Abnova), and/or rat anti-HA 1:100 ods used for vector integration site analysis is
with 5 ml of 25 mM TRIS/HCl buffer plus 50 mM (Roche Applied Science). Secondary antibodies reported in the supplementary materials, mate-
NaCl, pH 7.5, enzyme activity retained by the were incubated 1:500 for 1 hour at RT as follows: rials and methods section.
column was eluted by 2.5 ml TRIS/HCl buffer donkey anti–sheep Alexa488, donkey anti–goat
plus 250 mM NaCl, pH 7.5 (starting from frac- Alexa546, donkey anti–rat Alexa488, and donkey
tion n.5). Fractions (0.5 ml) were collected and anti–rabbit Alexa647. Nuclei were counterstained References and Notes
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Supplementary Materials
www.sciencemag.org/cgi/content/full/science.1233158/DC1
28. S. Groeschel et al., Metachromatic leukodystrophy: Development. Clin. Pediatr. (Phila.) 42, 427–432
Materials and Methods
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Figs. S1 to S22
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www.sciencemag.org SCIENCE VOL 341 23 AUGUST 2013 1233158-11

 Lentiviral Hematopoietic Stem Cell Gene Therapy Benefits
Metachromatic Leukodystrophy
Alessandra Biffi et al.
Science 341, (2013);
DOI: 10.1126/science.1233158

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