Lentiviral Hematopoietic Stem Cell Gene Therapy for MNGIE

The results presented in this PhD thesis demonstrate the efﬁcientand persistent correction of the biochemical phenotype in
affected organs of Tymp-/-Upp1-/- mice, brain neurologicalphenotypes (white matter edema and astrocytes morphology),
and intestinal myopathy (atrophy of the muscularis propria), at
relatively low vector copy numbers and without obvious vector ordisease-related phenotoxicity or genotoxicity. Due to the lack ofsome of the major neurological or gastrointestinal clinical-/-/phenotypes in Tymp Upp1 mice (such as motor dysfunction orcachexia), researchers should be cautious in translating our
ﬁndings from mice to MNGIE patients. Nonetheless, it isrecommended to perform more studies before clinicalapplication, including evaluating HSCGT in more clinicallyrelevant models that mimic human MNGIE disease, the dosing of
LV vector and transplanted gene modiﬁed cells, and a morethorough assessment of vector biodistribution and safety studies
addressing potential phenotoxicity and genotoxicity.

CGACTGCCGAGCTCCGCCCTCCAGGCGGCCCCACCCGCCTGCCGTCCTGGGGCGCCGCCGCCCCGCCGCCGGCAGTGGACCGCTGTGCGCGAACCCTGAACCCTACGGTCCCGACCCGCGGGCGAGGCCGGGTACCTGGGCTGGGATCCGGAGCAAGCGGGCGAGGGCAGCGCCCTAAGCAGGTA
CGGGCGGGGCTCAAGTCGCGAGGCGGGGAAGCGGGAGGCAGACACGGACGAGGGCGACACAGACACGGGACCGAGGGGCGGACACCGGAGAGACACGGGAAAGGGGTCGGGACAGGAGCACGTGGCTCAGACACCGACGCCGGGAGGCCGCAGACCCCGGACGTGTCAGGCA TCCCCGCAGGC
CCGGAGCGATGGCAGCCTTGATGACCCCGGGAACCGGGGCCCCACCCGCGCCTGGTGACTTCTCCGGGGAAGGGAGCCAGGGACTTCCCGACC
CTTCGCCAGAGCCCAAGCAGCTCCCGGAGCTGATCCGCATGAAGCGAGACGGAGGCCGCCTGAGCGAAGCGGACATCAGGGGCTTCGTGGCCG
CTGTGGTGAATGGGAGCGCGCAGGGCGCACAGATCGGTGCGTGGGGAGGGTTGGGCGTTCCTGACCCCGACTGGGAGGTCAGCCCGAGAGACT
TTGGGTCCCTGGGGGTGCGACGGTGCCCCACTACCAGCACCGGCCCCAGGGTGCCCCACCGCTGTGGGCTGCCACCCTCACGCGT ACCCCCAC
ATACCAGGGGCCATGCTGATGGCCATCCGACTTCGGGGCATGGATCTGGAGGAGACCTCGGTGCTGACCCAGGCCCTGGCTCAGTCGGGACAGC
AGCTGGAGTGGCCAGAGGCCTGGCGCCAGCAGCTTGTGGACAAGCATTCCACAGGGGGTGTGGGTGACAAGGTCAGCCTGGTCCTCGCACCTG
CCCTGGCGGCATGTGGCTGCAAGGTTAGAAACCACCTCCTTTCCAGACGGGAGCCTATACCGCACATGCAGCAACCAGTCCATCCACAGGCAGCTCCCAACCTCAAGCCTGGCCCAAAGCCTCCAAGACCCTACCAAGGCTTCTCCCCACCCTGCTCCCCAGCACAGTTCTCCCCACCCCGTTCCCCAGC
ACAGCGCTTGGGGCCCCTCTGGCTCCAGACCAGGCCCCTTGGAGCAGGAAAAAGATCCACTGATGGAATTCAGACCCCTTTCCCCTTGGGTCCCC
AGACAGCTCCCCCAAGGGAGGAGCTGAGGACTTTC CTCCCTCTGCCCCAAGCCTTGTTTCCCCAAGGACAGGTACCAACCTCCTCCCCTACTGAC
ACTTCTCAACCAAGAAAACTTCCTTTCCATTCCCTCACCAGCTGGGCACCCCTATAGCTGCTTAAATACTTTCCAAATCCAGCTGCACTCCTAGCCAG
GGAAGGTGAAGGGATGCACAGAGGTGGGGGAGGGGTACTGTGCAGGGTACTCAGCATCCCTGACCACCAGGTGCCAATGATCAGCGGACGTGGT
CTGGGGCACACAGGAGGCACCTTGGATAAGCTGGAGTCTATTCCTGGATTCAATGTCATCCAGAGCCCAGAGCAGGTACGGGGCGCCACGGATCAGTCATTAATCCAGGTTGATGATGGAGACCCTGGCCAGAATCACTAAAAGATCACTGGTGGATCATTAGGGTCACTAATGAGAACACTGGTCAAGGTTA
CTCATGAGTCACTGGGCCTGGGCCGAAATCATCAGTGGAACTTTGATTAGGATCATAAAATGGGAAGTTGGTCAAAATCACAGATGGCTGGCGGGG
CACGGTGGCTCACACCTGTAGTCCTAGCACTTGGGGAGGCCGAAGAGGGCAGATCCCTTGAACCCAGGAGTTCAAAACCAGCCTGGATAACACGGCAAAACCCCATCTCTACAAAATAGTTCGCTGCGTGTGGTGGTGCACGCATGTGGTTCCAGCTACTCAGGAGGCTGAGGCAGGAGGATCACTTGAGCCTGGGAGGTCTAGGCTGCAGTGAGCCGGGACGATGCCACTGCACTCCAGCCTGGGCAACAGAGTGAGACCCTGTCCCAGCACTCTGGGAGGCAGAGGAGCCCAGTTGGAGATCAGCCTGGGTAATATAGTGAAACTTGATCTCTACAAAAAAAAGAAGAAAAAAAAAAGCCGCGTGTGGTGGTGCGCAC
CTGTAGTCCCAGCTACTGGGAAGCTGAGGTGGGAGGATCACTTAAGCCCAGGAGGCAGAGGTCACAATGAGCCGAAATTGTGCCAACTGCACTCC
AGCCTGGGCAACAGAGGAAGACTCTTCACAGAAAAAAAAAAAAAAAAAAAAAAAGCTGCT AAGTCATTTACCATAAGTCACTGAGAACAGGGGATGTCTGACCAGATGCAAGTGCTGCTGGACCAGGCGGGCTGCTGTATCGTGGGTCAGAGTGAGCAGCTGGTTCCTGCGGACGGAATCCTATATGCAGCCAGAGATGTGACAGCCACCGTGGACAGCCTGCCACTCATCACAGGTGACCTGACTCCATGGCCTGCTTCTGCATGTTCACAGGCTCCTGACCTCCAAACTCAAGTCAAGGGCCTCTCGTTAGGAGTTACCCGTCACCTGACCGTGTGCCCCCCTACCCCCATCACAAGATGCCTGACCACCACCATGTGGGGT
GGCCTGATACTCAACCCACCAGGTGCTGCCACCCCCATAATAAGGGACTTGACCCTCAATGCTCAGGGCCCCTGACCCCAAAGTCGGCATCCCCGAACTCTCCCAAGAAGCTCCAGGTTCTCCATTGTCTCCAACCTCCTCTGCCTCCCCCAAAGCCTCCATTCTCAGTAAGAAACTCGTGGAGGGGCTGTCCGCTCTGGTGGTGGACGTTAAGTTCGGAGGGGCCGCCGTCTTCCCCAACCAGGAGCAGGCCCGGGAGCTGGCAAAGACGCTGGTGAGCGGTGT
GGCCTTTCCCTGGGCAAGCGTCTTGATGCGGGCCCAGCCTACCCTTCACCCCTCCCGTCCCCACTGCCTCCCTCCACTCAGCAGTCCTGCCT AAC
CCCAGTCCCACCCTCTTCTGCCCGAAGTCCCTCCCTCCTTCACGGCTTCCTAACCTGCTGTGACTTTAGAGGTCAAGGCTGGCCCGGCCTGGACCTGGGGAAGCCCTCTGTGGCGTTCCTGCCCCAGACCAAGTACAAGTTCCTCCTGGCCCCATGGCGAGGTGTCGCACTTCACTCGTGTCTCTTCCCC
ACCCCAATCCTTCCCTGACTTCATGCTGGGGGGCTGGCAACCCAGGGTGCAGCAGGGGCTGGAGTTCGACCAAGAACCGGCTGCAGAAGGCCCC
GCCATGGGGGGTCCACGCTGAGCCTCCTCTCCGCAGGTTGGCGTGGGAGCCAGCCTAGGGCTTCGGGTCGCGGCAGCGCTGACCGCCATGGAC
AAGCCCCTGGGTCGCTGCGTGGGCCACGCCCTGGAGGTGGAGGAGGCGCTGCTCTGCATGGACGGCGCAGGCCCGCCAGACTTAAGGGACCTG
GTCACCACGCTCGGTGAGGGGGACGGGGTGTAGGGGAGCGGAGGCGGCGGGGGGTGCTTCCCGCTGGGGCCGCCCCGACCCGGCCGCGCCTAAGACCCGTCCCCGCCCGCAGGGGGCGCCCTGCTCTGGCTCAGCGGACACGCGGGGACTCAGGCCCAGGGCGCTGCCCGGGTGGCCGCGGC
GCTGGACGACGGCTCGGCCCTTGGCCGCTTCGAGCGGATGCTGGCGGCGCAGGGCGTGGATCCCGGTCTGGCCCGAGCCCTGTGCTCGGGAAGTCCCGCAGAACGCCGGCAGCTGCTGCCTCGCGCCCGGGAGCAGGAGGAGCTGCTGGCGCCCGCAGA TGGTGAGCGTCGGGGGAGTCCCCGT
CCTTCCGCCTCCGCCATCCCCTTCCCTTCCCGAGGCCCCGCCCCTTCCCGAGCCCGCGCCTCTCAGCCCCTCTCCCCGCAGGCACCGTGGAGCTGGTCCGGGCGCTGCCGCTGGCGCTGGTGCTGCACGAGCTCGGGGCCGGGCGCAGCCGCGCTGGGGAGCCGCTCCGCCTGGGGGTGGGCGCAGAGCTGCTGGTCGACGTGGGTCAGAGGCTGCGCCGTGGTGAGCGCCGCCCCCGCCCTGCTGGCCCCGCACCCCCGCCCAGCTCCGGCCGCGC
GGCCTCTAACAGCCCCTCGCTCTGCAGGGACCCCCTGGCTCCGCGTGCACCGGGACGGCCCCGCGCTCAGCGGCCCGCAGAGCCGCGCCCTG
CAGGAGGCGCTCGTACTCTCCGACCGCGCGCCATTCGCCGCCCCCTCGCCCTTCGCAGAGCTCGTTCTGCCGCCGCAGCAATAAAGCTCCTTTGC
CGCGAAAGGGCCGGGCGCAGCCGCGCTGGGGAGCCGCTCCGCCTGGGGGTGGGCGCAGAGCTGCTGGTCGACGTGGGTCAGAGGCTGCGCC
GTGGTGAGCGCCGCCCCCGCCCTGCTGGCCCCGCACCCCCGCCCAGCTCCGGCCGCGCGGCCTCTAACAGCCCCTCGCTCTGCAGGGACCCC
CTGGCTCCGCGTGCACCGGGACGGCCCCGCGCTCAGCGGCCCGCAGAGCCGCGCCCTGCAGGAGGCGCTCGTACTCTCCGACCGCGCGCCATTCCCGGGACGGCCCCGCGCTCAGCGGCCCGCAGAGCCGCGCCCTGCAGGAGGCGCTCGT ACTCTCCGACCGCGCGCCATTCTCCCTTCGCAGAGCTCGTTCTGCCGCCGCAGCAATAAAGCTCCTTTGCCGCGAAAGGGCCGGGCGCAGCCGCGCTGGGGTGCGGGCGCAGCCGCGCTGGGGTGCTGCAGGGACCCCCTGGCTCCGCGTGCACCGGGACGGCCCCGCGCTCAGCGGCCCGCAGAGCCGCGCCCTGCAGGAGGCGCTCGT ACTCTCCGACCGCGCGCCATTCCCGGGACGGCCCCGCGCTCAGCGGCCCGCAGAGCCGCGCCCTGCAGGAGGCGCTCGTACTCTCCGACCGCGCGCCATTCT
CCCTTCGCAGAGCTCGTTCTGCCGCCGCAGCAATAAAGCTCCTTTGCCGCGAAAGGGCCGGGCGCAGCCGCGCTGGGGTGCGGGCGCAGCCGC
GCTGGGGTGCTGCAGGGACCCCCTGGCTCCGCGTGCACCGGGACGGCCCCGCGCTCAGCGGCCCGCAGAGCCGCGCCCTGCAGGAGGCGCTCGTACTCTCCGACCGCGCGCCATTCCCGGGACGGCCCCGCGCTCAGCGGCCCGCAGAGCCGCGCCCTGCAGGCGCCGCAGCAA TAAAGCTCCT
TTGCCGCGAAAGGGCCGGGCGCAGCCGCGCTGGGGTGCGGGCGCAGCCGCGCTGGGGTGCTGCAGGGACCCCCTGGCTCCGCGTGCACCGG
GACGGCCCCGCGCTCAGCGGCCCGCAGAGCCGCGCCCTGCAGGAGGCGCTCGT ACTCTCCGACCGCGCGCCATTCCCGGGACGGCCCCGCGC
TCAGCGGCCCGCAGAGCCGCGCCCTGCAGGAGGCGCTCGTACTCTCCGACCGCGCGCCATTCTCCCTTCGCAGAGCTCGTTCTGCCGCCGCAG
CAATAAAGCTCCTTTGCCGCGAAAGGGCCGGGCGCAGCCGCGCTGGGGTGCGGGCGCAGCCGCGCTGGGGTGCTGCAGGGACCCCCTGGCTCCGCGTGCACCGGGACGGCCCCGCGCTCAGCGGCCCGCAGAGCCGCGCCCTGCAGGAGGCGCTCGT ACTCTCCGACCGCGCGCCATTCCCGGG
ACGGCCCCGCGCTCAGCGGCCCGCAGAGCCGCGCCCTGCAGGGCAGAGCCGCGCCCTGCAGGAGGCGCTCGT ACTCTCCGACCGCGCGCCATTCCCGGGACGGCCCCGCGCTCAGCGGCCCGCAGAGCCGCGCCCTGCAGGAGGCGCTCGT ACTCTCCGACCGCGCGCCATTCTCCCTTCGCAGAGCTCGTTCTGCCGCCGCAGCAATAAAGCTCCTTTGCCGCGAAAGGGCCGGGCGCAGCCGCGCTGGGGTGCGGGCGCAGCCGCGCTGGGGTGCTGCAGGGACCCCCTGGCTCCGCGTGCACCGGGACGGCCCCGCGCTCAGCGGCCCGCAGAGCCGCGCCCTGCAGGAGGCGCTCGT ACTCTCCGACCGCGCGCCATTCCCGGGACGGCCCCGCGCTCAGCGGCCCGCAGAGCCGCGCCCTGCAGGCGCGCCCTGCAGGAGGCGCTCGT ACTCTCCGA

Lentiviral Hematopoietic Stem Cell
Gene Therapy for MNGIE

Rana Yadak
2017

Rana Yadak

Lentiviral Hematopoietic Stem Cell Gene Therapy for MNGIE
Lentivirale hematopoietische stamcelgentherapie voor MNGIE

Rana Mahmoud AbdulRaheem Yadak

ISBN: (provided by O

ISBN: 978-94-6182-857-6

Cover design: Rana

Cover design: Rana Yadak, the traditional Palestinian embroidery pattern “wide open eye [ ]”assembled as nucleobases in the DNA ladder, with a background of the human TYMP gene sequence.
gene sequence.
Layout and printing: Off Page, Amsterdam

Layout and printing

Acknowledgements

Copyright © Rana Y
The research in this thesis was financially supported by the European Commission’s 5th, 6th and 7thrights reserved. N
Framework Programs, CONSERT, CELL-PID, PERSIST , and by the Netherlands Health Research All
andsystem or transmitte
Development Organization ZonMw, Join4energy, Ride4Kids, and the Sophia Foundation (SSW0645)
when appropriate, oand Stichting NeMo.

Copyright © Rana Yadak, Rotterdam 2017

Acknowledgements

All rights reserved. No part of this thesis may be reproduced, distributed, stored in a retrieval system
The research in this th
or transmitted in any form or by any means without permission of the author, or, when appropriate,
Framework Programs,of the publishers of the publications.
and Development Org(SSW0645) and Sticht

Lentiviral Hematopoietic Stem Cell Gene Therapy for MNGIE
Lentivirale hematopoietische stamcelgentherapie voor MNGIE
Proefschrift
ter verkrijging van de graad van doctor aan deErasmus Universiteit Rotterdam
op gezag van derector magnificus

Prof.dr. H.A.P. Polsen volgens besluit van het College voor Promoties.
De openbare verdediging zal plaatsvinden op
dinsdag 19 december 2017 om 11:30 uur
door
Rana Mahmoud AbdulRaheem Yadak
geboren te Nablus, de West Bank, Palestina

Promotiecommissie:
Promotor:
				Prof.dr. P.A.E. Sillevis Smitt

Overige leden:
				Prof.dr. A.G. Vulto
				Prof.dr. J.J. Cornelissen				Prof.dr. R.C. Hoeben

Copromotoren:
				Dr. N.P. van Til
				Dr. I.F.M. de Coo‫إهداء للوالد و الوالدة‬For my father and mother.

| |Table of Contents
Chapter 1

General introduction

9

Chapter 2

Mitochondrial Neurogastrointestinal Encephalomyopathy 
Caused by Thymidine Phosphorylase Enzyme Deficiency:
From Pathogenesis to Emerging Therapeutic Options

17

Chapter 3

Development of a highly efficient lentiviral gene transfer protocol for 
hematopoietic stem cells

49

Chapter 4

Preclinical efficacy and safety evaluation of hematopoietic stem cell gene therapy in a mouse model of MNGIE

71

Chapter 5

Transplantation, gene therapy and intestinal pathology in 
MNGIE patients and mice

105

Chapter 6

Hematopoietic stem cell lentiviral integration profiles are stable across  117
multiple species, disease phenotypes and hematopoietic cell types

Chapter 7

General discussion

Appendix

Summary167
Samenvatting (Dutch summary)
169
Arabic summary (‫)ملخص‬173
List of terms and abbreviations175
PhD Portfolio 
177
List of publications
179
Curriculum vitae
180
Acknowledgements 
181

151

General introduction

1General introduction| |Hematopoietic stem cell gene therapy (HSCGT)
Hematopoietic stem cells (HSCs) are
multipotent stem cells with dual functional
attributes of self-renewal to retain
the stem cell pool and to differentiate into
cells of the immune system and blood
through hematopoiesis.1,2 The bone marrow(BM) niche is the microenvironment wherequiescent HSCs reside, hematopoiesis
occurs, and from which HSCs mobilize into
the periphery and home to and engraft after
BM transplantation. Primary stromal cells are
involved in these processes, in which adhesion
molecules, chemokines, and cytokines
play a major role (reviewed in Lapidot T
et al. 2002, 2005).3,4 This forms the basisof hematopoietic stem cell transplantation(HSCT). HSCT is a common clinicalprocedure to reconstitute the hematopoietic/
immune system after conditioning byradiation or chemotherapeutic regimens.
Allogeneic HSCT involves the transplantationof donor matched-BM, mobilized peripheral
blood, or umbilical cord blood (UCB)derived HSCs, and is the therapy of choice fora variety of (non-) malignant hematologicaland non-hematological conditions.5 Graftversus host disease, graft failure, and toxicityof the preparative regimens are common
complications related to allogeneic HSCT.
Autologous hematopoietic stem cell genetherapy (HSCGT), the ex vivo transfer ofa functional copy of a gene (therapeuticgene) into the patient’s own hematopoietic
stem/progenitor cells (HSPCs), is anotherapplication of HSCT.
The family of retroviruses contains two
members that form the basis of viral vectors
used in clinical trials: i.e. gammaretrovirus(γ-RV) and lentivirus (LV), which have been1

successfully used for HSCGT due to theirability to permanently integrate their DNA
into the host HSPCs’ genomic DNA. LV vectors
offer several advantages over γ-RV vectors.
First, LVs possess a central polypurine tract(cPPT) element which significantly improves
nuclearimportofthepre-integration

complex, therefore LV vectors can transduce
6

non-dividing quiescent HSCs. The capacity
to transduce non-dividing HSCs significantly
reduces the need for excessive ex vivo HSPCs
cytokine stimulation to proliferate comparedwith γ-RV vectors, which is detrimental forthe long term repopulation of gene modified
HSCs in vivo.7 Second, insertional oncogenesis
has been observed in clinical trials for
X-linked SCID, Wiskott-Aldrich syndrome,and chronic granulomatous disease using
γ-RV.8-12 In contrast, animal studies showedthat the LV integration profiles are relatively
safe compared to γ-RV as demonstrated
with adrenoleukodystrophy.13 Due to theseadvantages,

HSCGT

has

recently

been

applied successfully in several hematological,
immunological and metabolic disorders.14
The rationale for using HSCGT to target
metabolic diseases relies on the capacity of
the gene modified HSPCs and their progeny
to act as a factory of the therapeutic proteins.
The protein’s substrate can be converted in
the gene modified cells or the protein excretedanddeliveredthroughcross-correction.

HSCs’ self-renewal capacity and the highly
proliferative progenitors mediate the long
lasting effects of HSCGT. The rationale forand the clinical procedure of autologous
LV-based HSCGT are presented in Figure 2A,
Chapter 2.

11Chapter 1| |Mitochondrial neurogastrointestinal
encephalomyopathy (MNGIE)MNGIE is a rare metabolic disease caused bymutations in the TYMP gene, which resultsin defective thymidine phosphorylase (TP)enzyme activity and subsequent increase in its
nucleoside substrates, deoxyribonucleosides
thymidine(dThd)
anddeoxyuridine(dUrd). This consequently results in alteredmitochondrial DNA (mtDNA). Due tothe limited therapeutic efficacy of the available
treatments and the severe toxicity related

12

to allogeneic HSCT, gene therapy emerged
as an attractive alternative for treatment of
MNGIE. The experiments performed in thisthesis were designed to evaluate the efficiency
and safety of LV based HSCGT for MNGIE.
Genetic defects, clinical manifestations,diagnosis, pathogenesis, current treatments,
pre-clinical studies, and future directions in
MNGIE research are discussed in Chapter 2.

Scope and outline of this thesis| |Scope and outline of this thesis
To cure rare inherited diseases, HSCGT has
emerged as a promising platform for numerous
hematological, immunological, and metabolic
disorders1 (reviewed in Naldini, 2015).14
The currently investigated treatment optionsfor MNGIE often only provide temporary
restoration of biochemical homeostasis thatlacks long-term improvement of clinicalphenotypes, such as infusion of platelets andperitoneal dialysis. Allogeneic HSCT could bea potential curative treatment, but is associatedwith substantial morbidity and mortality(discussed in Chapter 2). A recent approachto performing liver transplantation has
obvious drawbacks, such as life-long immune
suppression and graft failure. Alternative
therapies that provide life-long effective
biochemical and phenotypic correction
with acceptable adverse effects are an urgent
medical need. The studies performed in thisthesis were designed to evaluate the potential
application of LV based HSCGT as a safe andefficient treatment for MNGIE.

(i) Optimization of culture
conditions for highly efficient
LV transduction of HSPCs
without compromising long term
repopulation efficiency and genemarking in vivo.
Most HSPC transduction protocols use
multiple rounds of LV vector incubations withhigh vector doses, yielding many integrated
LV copies per genome, and as a consequence,a relatively high genotoxicity risk. Also,

1

1high concentrations of multiple cytokines
are used to induce HSPC proliferation andenhance transduction efficiency. Therefore,
the development of highly efficient LVtransduction protocols for HSPCs arenecessary. Optimizing parameters such as
the duration of the transduction, reducing
the number of integrations per cell, andrestriction of growth factors, would increase
the number of patients that could be treatedper LV vector stock, thereby reducing
the costs substantially. Furthermore, this
could potentially also improve long-term
therapeutic outcomes. To address these points,
the following experiments were designed
as described in Chapter 3. First, lineagedepleted (Lin-) mouse BM cells, rhesus BM
CD34+ cells, and human peripheral blood and
BM CD34+ progenitor cells, were transduced
overnight at an increasing cell density witha proportional increase of transducing units
in order to increase their physical contact.
Second, lin- cells and human umbilicalcord blood CD34+ cells were transduced
overnight under varying concentrations of
selected growth factors either singularly or in
combinations. The repopulation capacity andstable gene marking in vivo was assessed by
the transplantation of lin- cells into sublethally
irradiated wild type recipient mice.

(ii) Efficiency assessment of LV based
HSCGT for MNGIE
Initial attempts demonstrated correctionof the biochemical phenotype in a mousehttps://clinicaltrials.gov

13

Chapter 1

model of MNGIE following LV based
HSCGT,15 encouraging its further pre-clinicaldevelopment as a treatment option for
MNGIE. We aimed at addressing the efficiency
in depth, particularly on neurological andintestinal phenotypes which are otherwise
unresponsive to allogeneic HSCT. We tested
third generation self-inactivating LV vectors
with a backbone similar to those applied inclinical trials with a modified Woodchuckpost-transcriptional regulatory element,
and the cellular human phosphoglycerate
kinase (hPGK) promoter to drive the human(TYMP) or codon optimized (TYMPco)
cDNA sequences (Figure 1A, Chapter 4).
The efficacy of HSCGT was tested in Tymp/Upp1-/- for up to 8 - 11 months. Dose
scaling of LV vectors and transplanted genemodified cells was performed to establish
the minimum required VCN/cell sufficientfor biochemical correction. The evaluationof therapeutic outcomes (Chapter 4)
involved assessments of (a) molecular
chimerism and integrated LV vector copies;(b) biochemical phenotype in urine, blood,
brain, intestine, liver, and muscle tissues;(c) neurological phenotypes by performing
memory and motor function assessments,

14

brain MRI and immunohistochemistry; andin Chapter 5 (d) we assessed the pathology ofthe small intestine of mice receiving HSCGT
as well as MNGIE patients treated withallogeneic HSCT.

(iii) Safety assessment of LV based
HSCGT for MNGIE
To address any potential side effects related to
TP overexpression or LV- related genotoxicity,
we performed experiments as described in
Chapters 4 and 6: (a) Tymp-/-Upp1-/- micetransplanted with gene modified HSPCsbearing LV vectors with a strong promoter/enhancer derived from spleen focus forming
virus (LV-SF) were monitored long term.
(b) Long term follow up was performed on
Tymp-/-Upp1-/- mice recipients of HSPCs
transduced with a therapeutic vector
containing the PGK promoter. Both under(a) and (b) the discomfort was monitored
and hematological analysis was performed
to exclude hematological abnormalities. (c)Secondary transplantations were performed
to assess tumor incidence up to 11 months.
(d) LV integration sites were retrieved by
LAM-PCR on genomic DNA of lin- cells andin BM cells after transplantation.

Scope and outline of this thesis| |References
1.

Doulatov S, Notta F, Laurenti E, Dick JE.
Hematopoiesis: a human perspective. Cell
Stem Cell. 2012;10:120-136.

2.

Ema H, Kobayashi T, Nakauchi H. Principles
of Hematopoietic Stem Cell Biology. In: Kondo
M, ed. Hematopoietic Stem Cell Biology.
Totowa, NJ: Humana Press; 2010:1-36.

3.

Lapidot T, Petit I. Current understandingof stem cell mobilization: the roles ofchemokines, proteolytic enzymes, adhesion
molecules, cytokines, and stromal cells. Exp
Hematol. 2002;30:973-981.

4.

Lapidot T, Dar A, Kollet O. How do stem cells findtheir way home? Blood. 2005;106:1901-1910.

5.

Copelan
EA.
Hematopoietic
stem-cell transplantation. N Engl J
Med. 2006;354:1813-1826.

6.

Follenzi A, Ailles LE, Bakovic S, Geuna M, Naldini
L. Gene transfer by lentiviral vectors is limited by
nuclear translocation and rescued by HIV-1 pol
sequences. Nat Genet. 2000;25:217-222.

7.

Miller DG, Adam MA, Miller AD. Genetransfer by retrovirus vectors occurs only incells that are actively replicating at the time ofinfection. Mol Cell Biol. 1990;10:4239-4242.

8.

Hacein-Bey-Abina S, von Kalle C, Schmidt
M, et al. A serious adverse event after
successful gene therapy for X-linked severe
combined immunodeficiency. N Engl J
Med. 2003;348:255-256.

9.

Hacein-Bey-Abina S, Von Kalle C,
Schmidt M, et al. LMO2-associatedclonal T cell proliferation in twopatients after gene therapy for SCID-X1.
Science. 2003;302:415-419.

10.

Hacein-Bey-Abina S, Garrigue A, Wang GP,
et al. Insertional oncogenesis in 4 patients
after retrovirus-mediated gene therapy of
SCID-X1. J Clin Invest. 2008;118:3132-3142.

11.

Braun CJ, Boztug K, Paruzynski A, et al.
Gene therapy for Wiskott-Aldrich syndrome-long-term efficacy and genotoxicity. Sci
Transl Med. 2014;6:227ra233.

12.

Stein S, Ott MG, Schultze-Strasser S, et al.
Genomic instability and myelodysplasia withmonosomy 7 consequent to EVI1 activation
after gene therapy for chronic granulomatous
disease. Nat Med. 2010;16:198-204.

13.

Biffi A, Bartolomae CC, Cesana D, et al.
Lentiviral vector common integration sites inpreclinical models and a clinical trial reflect
a benign integration bias and not oncogenic
selection. Blood. 2011;117:5332-5339.

14.

Naldini L. Gene therapy returns to centre
stage. Nature. 2015;526:351-360.

15.

Torres-Torronteras J, Gomez A, Eixarch H,
et al. Hematopoietic gene therapy restoresthymidine phosphorylase activity in a cell
culture and a murine model of MNGIE. Gene
Ther. 2011;18:795-806.

1

15

Mitochondrial Neurogastrointestinal
Encephalomyopathy Caused by Thymidine
Phosphorylase Enzyme Deficiency: From
Pathogenesis to Emerging Therapeutic Options
Rana Yadak1, Peter Sillevis Smitt1, Marike W. van Gisbergen2,
Niek P. van Til3, Irenaeus F. M. de Coo1
Department of Neurology, Erasmus University Medical Center, Rotterdam, The Netherlands;
2
Department of Radiation Oncology (MaastRO-Lab), GROW – School for Oncology andDevelopmental Biology, Maastricht University Medical Centre, Maastricht, The Netherlands;
3
Laboratory of Tumor Immunology, University Medical Center Utrecht, Utrecht, The Netherlands
1

Frontiers in Cellular Neuroscience. 2017; 11: 31. PMID 28261062

2

Chapter 2| |Abstract
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a progressive metabolic
disorder caused by thymidine phosphorylase (TP) enzyme deficiency. The lack of TP resultsin systemic accumulation of deoxyribonucleosides thymidine (dThd) and deoxyuridine(dUrd). In these patients, clinical features include mental regression, ophthalmoplegia andfatal gastrointestinal complications. The accumulation of nucleosides also causes imbalancesin mitochondrial DNA (mtDNA) deoxyribonucleoside triphosphates (dNTPs), which may playa direct or indirect role in the mtDNA depletion/deletion abnormalities, although the exact
underlying mechanism remains unknown. The available therapeutic approaches include dialysis
and enzyme replacement therapy, both can only transiently reverse the biochemical imbalance.
Allogeneic hematopoietic stem cell transplantation (AHSCT) is shown to be able to restore
normal enzyme activity and improve clinical manifestations in MNGIE patients. However,transplant related complications and disease progression result in a high mortality rate. New
therapeutic approaches, such as adeno-associated viral (AAV) vector and hematopoietic stem
cell gene therapy have been tested in Tymp-/-Upp1-/- mice, a murine model for MNGIE.
This review provides background information on disease manifestations of MNGIE
with a focus on current management and treatment options. It also outlines the pre-clinicalapproaches towards future treatment of the disease.
Key words: mitochondrial neurogastrointestinal encephalomyopathy; MNGIE; thymidine
phosphorylase; metabolic disease; HSCT; HSCGT; lentiviral vector

18

Pathogenesis, current and prospective treatments for MNGIE| |Introduction
Mitochondrial
diseases
representa genetically and clinically heterogeneousgroup of disorders caused by mutations inmitochondrial DNA (mtDNA), that affectsynthesis and function of mitochondrial
proteins, such as tRNA (in MELAS disease)and ND1, 4, 6 (responsible for the majorityof cases in LHON disease) (DiMauro, 2004).
Another group is caused by mutations in
nuclear DNA (nDNA) that lead to defects
in nuclear encoded mitochondrial proteins.
Part of these proteins exert their effect on
mtDNA maintenance, thus known as nuclearmitochondrial communication disorders.
A subtype of the latter is mitochondrial
DNA depletion syndrome (MDS); a groupof mainly autosomal recessive disorders
caused by defects in nuclear genes involved
in mtDNA replication (e.g. POLG and PEO1
causing hepatocerebal MDS), or genes crucial

for maintenance of mtDNA including TK2(responsible for myopathic MDS), RRM2B(encephalomypathic MDS) and thymidine
phosphorylase(TYMP)

gene2

mutationsassociated with MNGIE (El-Hattab and
Scaglia, 2013). MNGIE, initially describedin 1976 by Okamura (Okamura et al., 1976)
is a fatal rare inherited metabolic disorderwithout genetic or ethnic predisposition(Gamez et al., 2005). The estimated rate ofoccurrence is 1-9:1,000,0001, and as of 2011
fewer than 200 cases have been described in
the medical literature (Halter et al., 2011). Dueto its variable clinical presentations, MNGIE
can be easily overlooked or misdiagnosed
as Crohn’s disease, psychiatric disorder,anorexia nervosa or myasthenia gravis(Rickards et al., 1994;Teitelbaum et al., 2002;
Marti et al., 2004).

| |Genetic defects, clinical manifestations anddiagnosis
Genetic defects
MNGIE is an autosomal recessive inherited
disease that is caused by mutations in
the nuclear gene TYMP (previously known
as ECGF1). TYMP codes for the TP enzyme(EC 2.4.2.4.) and is located on chromosome
22q13.33 (Stenman et al., 1992).TP isa cytoplasmic enzyme expressed in mosthuman tissues, including gastrointestinal
tract, central and peripheral nervous
system, spleen, liver, bladder, leukocytes
and in platelets which account for most of
the TP activity in human blood (Fukami

1and Salganicoff, 1973;Shaw et al., 1988).
In contrast, TP is present at low levels inmuscles and is lacking in kidney, aorta
and fat tissues (Fox et al., 1995;Valentino
et al., 2007). MNGIE is caused by a varietyof pathogenic homozygous or compound
heterozygous mutations in the exons or
flanking regions of the TYMP gene. Various
mutations are reported to date (Stenson et al.,2014) including deletions, single nucleotide
insertions (Nishino et al., 1999), splice site
(Kocaefe et al., 2003;Szigeti et al., 2004b) andframeshift mutations (Blazquez et al., 2005)

http://www.orpha.net/consor/cgi-bin/index.php

19Chapter 2

and a homozygous duplication mutation inexon 8 of the TYMP gene (Gamez et al., 2005).
The majority of these mutations are loss offunction mutations. Heterozygous mutation
carriers are asymptomatic with approximately
35% residual TP activity, although the plasma
nucleoside levels are similar to healthy
controls (Marti et al., 2004).
Inaddition,
non-pathogenic
polymorphisms have been described in
the TYMP gene. The A465T polymorphism(c.1393G>A) was reported both in subjects
with MNGIE like features and control
subjects (Vissing et al., 2002;Martin et al.,2004). In some MNGIE cases there is no or
mild clinical involvement of gastrointestinal
tract or skeletal muscle, despite the presenceof mutations in the TYMP gene leading tomarked reduction in TP activity, probably
indicating that environmental factors
contribute to the severity of the clinicalsymptoms (Martin et al., 2004;Szigeti etal., 2004b). Apart from late-onset forms ofthe disease (Marti et al., 2005;Massa et al.,2009;Etienne et al., 2012), most patientsdisplay typical MNGIE features before the ageof 20 years (Nishino et al., 2000;Teitelbaum
et al., 2002).

Clinical manifestations
Gastrointestinal and ocular involvement areusually the first complications in this disease,although neuropathy and hearing loss havebeen reported as primary symptoms in somecases (Garone et al., 2011). Clinical symptoms
are summarized in Table 1.

Diagnosis
Detailed patient history, thorough clinicalexamination, particular findings on magnetic
resonance imaging (MRI) of the brain(Figure 1), genomic DNA screening formutations in TYMP gene and biochemical
analysis all contribute to the diagnosis of
MNGIE. Biochemical diagnosis of MNGIE
includes at least one of the following
parameters (Marti et al., 2004): (1) Increased
blood plasma levels of dThd and dUrd(>3µmol/L and >5µmol/L respectively). (2)Severely reduced TP enzyme activity in buffy
coat leukocytes (<8% of healthy controls;
healthy control mean TP activity equivalent
to 634 nmol thymine formed /hr/mg protein).
Biochemical analysis reduces the risk of
missing the diagnosis in case of non-identified
mutation sites (Nishino et al., 2000) or in caseof unclassified variants (UV). Additionally,
biochemical diagnosis contributes tothe confirmation or exclusion of the the roleof a UV as a cause for MNGIE. Similarly,
biochemical assessment is preferred overclinical diagnosis since some of the classicalsymptoms of MNGIE can be absent. Other
frequently observed findings in MNGIE
patients include metabolic abnormalities suchas lactic acidosis, deficiency of mitochondrial
respiratory chain enzymes, mainly complex I
and IV (Hirano et al., 1994) (Debouverie et al.,1997), urinary Thd and dUrd accumulation(Fairbanks et al., 2002;Spinazzola et al., 2002;laMarca et al., 2006) and elevated protein levels
in CSF (Bedlack et al., 2004). Infrequently,
skeletal muscle biopsies may reveal ragged
red fibers, and mtDNA analysis may reveal
acquired deletions, depletions or point
mutations (Teitelbaum et al., 2002;Nishigaki
et al., 2003).

20

Pathogenesis, current and prospective treatments for MNGIE

2

Figure 1. Brain MRI findings in MNGIE. MRI of MNGIE patient at age 16 with “ typical” MNGIE
phenotype . (A) T1 weighted sagital image shows cerebellar vermis atrophy (arrow) and normal
gyral pattern. (B) Axial T2 with hyperintensities in the dorsal pons and mesencephalon (arrow). (Ccoronal flair image, D axial T2) show extensive signal abnormalities in the cerebral white matter.
The external capsule is involved as is the inner blade of the corpus callosum (arrow C, D). (E, F)
Extensive white matter involvement with sparing of the U-fibers (arrow).

| |Pathogenesis
The TP enzyme converts mitochondrial
dThd and dUrd to the nucleotide bases
thymine and uridine respectively and 2-deoxy
ribose 1-phosphate (Friedkin and Roberts,
1954). This occurs in de novo synthesis or
via the salvage pathway. dThd and dUrd
are homogeneously present in cellular andplasma compartments and they translocate
between compartments through nucleosidetransporters (NTs). In humans two unrelated
protein families have been described (Younget al., 2013), concentrative nucleosidetransporters (CNTs), an active transportsystem, and equilibrative nucleosides
transporters (ENTs) responsible for passive
facilitated diffusion.
The bidirectional ENTs, mainly ENT1,
are ubiquitously present on almost all cell
types and mediate the uptake and efflux ofnucleosides (Figure 2B). Therefore they areimportant for cells that rely on the salvage
pathway for supply of nucleosides, includingbone marrow cells, erythrocytes andleukocytes, brain and muscles (Young etal., 2008). Although TP is not expressed in
all tissues, the TP expressed in circulating
21

Chapter 2
Table 1. Common and rare clinical symptoms in MNGIE patients.
Complication

Symptoms

Gastrointestinal Appetite loss, satiety
Weight loss
Digestive features: Chronic
diarrhea, abdominal pain,
cramps, nausea, colonic
distension, dysphagia
OcularAuditory

External ophthalmoplegia,
ptosis, retinal pigmentarychanges, glaucoma, optic
nerve atrophy
Deafness

Pathophysiology
Myogenic (visceral smooth muscle):
atrophy in the muscularis propria of the stomach andsmall intestines.
Neurogenic (enteric nervous system): loss of
the interstitial cells of Cajal.
Mixed myo-neurogenic causes

Dysfunction of cranial nerve and auditory cortex.
Atrophy of the stria vascularis in the cochlea

CNS

Mental changes, subcorticalloss of cognitive functions,memory impairment

leukencephalopathy

PNPs

Numbness and paraesthesia

Demyelinating sensorimotor type: reduced sensory
motor conduction, loss of myelin sheaths in lumbar
and brachial plexus.

Skeletal muscle Proximal myopathy

OthersmtDNA molecular alterations and abnormal respiratory
chain enzymes in skeletal muscles

Endocarditis
Spontaneous abdominal
esophageal perforation
Short stature
Cardiomyopathy
Psoriasis

CIPO, Chronic intestinal pseudo obstruction; CPEO, Chronic progressive external ophthalmoplegia;
CNS, central nervous system; PNPs, polyneuropathies

22

Pathogenesis, current and prospective treatments for MNGIE

RemarksStudy

A major cause of death and survival is generally related
to the severity of these symptoms.

(Granero Castro et al., 2010),(Garone et al.,2011),(Giordano et al., 2008),(Zimmer et al.,2009) ,(Perez-Atayde et al., 1998) ,(Blondon etal., 2005),(Chapman et al., 2014).

Can lead to severe denutrition, anaemia and eventually
the necessity for nutritional supportive treatments.

2

CIPO in the early disease course is under recognized.
CPEO phenotype is often present. Recovered upon HSCT (Threlkeld et al., 1992),(Barboni et al.,transplantation compared to untreated patient.
2004),(Vinciguerra et al., 2015)

Hearing loss is common among patients(in 61% of patients).

(Hirano et al., 1994), (Yasumura et al., 2003),(Li et al., 2011), (Mattman et al., 2011)

Satisfactory results were obtained soon following
cochlear implantation in MNGIE patients.
MNGIE is an example of an adult mitochondrial disorderin which leukodystrophy is observed.

(Millar et al., 2004),(Barragan-Campos et al.,2005),(Scaglia et al., 2005) ,(Schupbach et al.,2007),(Schiffmann and van der Knaap, 2009)
Patients presenting the characteristic multisystem
symptoms of MNGIE have a unique pattern on brain MRI ,(Scarpelli et al., 2013),(Carod-Artal et al.,indicative of vasogenic oedema and glial cell dysfunction. 2007;Salsano et al., 2013)
To date, it is debatable whether or not the extent ofthese brain MRI signal alterations, correlates with age,clinical severity, CNS involvement or the biochemical andgenetic profiles of MNGIE patients.
Neuropathy usually is not among the first symptoms of(Simon et al., 1990),(Hirano et al.,the disease.
1994),(Bedlack et al., 2004),(Menezes andOuvrier, 2012),(Pupe et al., 2012)
Some MNGIE cases are misdiagnosed with chronic
inflammatory demyelinating polyneuropathy.
Two cases with classical clinical presentation of MNGIE, (Papadimitriou et al., 1998),(Hirano et al., 2004),
were reported without skeletal muscle involvement.
(Szigeti et al., 2004b;Cardaioli et al., 2010;
Both cases showed identical homozygous splice-acceptor Bax et al., 2013)site mutation in TYMP gene (c.215-1G>C), which may
suggest a genotype-phenotype correlation.
Rare complications(Hirano et al., 1994),(Yolcu et al.,Short stature as seen in many mitochondrial diseases and 2014),(Kalkan et al., 2015)
partly as a complication of failure to thrive23Chapter 2

Figure 2. Schematic representation of autologous hematopoietic stem cell based genetherapy for MNGIE and possible mechanism of biochemical correction by gene modified
HSCs. (A) Autologous bone marrow (BM) aspirates or apheresis of peripheral blood HSCs (PBSCs)
after treatment with rh-G-CSF or plerixafor are collected from MNGIE patient. HSCs are ex vivotransduced by GMP grade lentiviral vectors containing the human TYMP transgene. Before infusionof the transduced cells, MNGIE patients are pre-treated with non-myeloablative conditioning toallow minimal engraftment of gene modified HSCs. (Selection and ex vivo expansion of genemodified HSCs allows for transplantation of large numbers of gene modified HSCs to obviate
the need for myeloablative pre-conditioning and allows (to some degree) for assessment of safety of
the gene modified HSCs prior to transplantation, for example by lentiviral vector integration analysis(reviewed in (Watts et al., 2011). (B) The enzyme thymidine phosphorylase (TP) is deficient in all
tissues of MNGIE patients, which leads to accumulation of the nucleoside substrates dThd and dUrd
and depletion of the nucleotide dCTP and finally mtDNA depletion and deletion (Gonzalez-Vioque
et al., 2011). Following transplantation of gene modified HSCs and homing to bone marrow, thesecells differentiate into all types of blood cells, LV genome and human TYMP transgene are integrated
in leukocyte DNA ensuring stable expression of TP. TP catalyzes the chemical reaction which
breaks down the nucleosides. This process eventually leads to reduction of systemic nucleosides
accumulation. Nucleoside transporters mediate nucleosides transfer via passive facilitated diffusion(ENTs) and active transport (CNTs), the ubiquitous bidirectional ENTs are depicted (Young et al.,2013). In addition, some gene modified HSCs differentiate into monocytes and may migrate to
the brain giving rise to microglia which act as a TP reservoir and cross correct the other cells in CNS.

24

Pathogenesis, current and prospective treatments for MNGIE

platelets and leukocytes and some othertissues is essential to degrade the excess
amounts of dThd and dUrd nucleosides which
are secreted into the blood (Lara et al., 2007).
The molecular pathological mechanism in
MNGIE involves imbalanced nucleosides andnucleotide pools. Initially, loss of function
mutations in TYMP gene were identified
resulting in reduced TP activity (Nishino etal., 1999) leading to accumulation of accessamounts of the nucleoside substrates inblood plasma, urine and almost all tissues(Spinazzola et al., 2002;Valentino et al., 2007).
It has been hypothesized that this biochemical
imbalance disturbs the equilibrium of
intra-mitochondrial
deoxyribonucleoside
triphosphates (dNTPs) pools (Spinazzola etal., 2002) and hence is responsible for mtDNA
depletion, multiple deletions and point
mutations associated with MNGIE (Hirano
et al., 1994;Papadimitriou et al., 1998)(Nishino et al., 2000;Nishigaki et al., 2003).
Therefore, recent studies have addressed
the relationship between biochemical anddNTP pool imbalances and subsequent
mtDNA abnormalities in MNGIE. In vitro,
mtDNA point mutations and deletions,similar to those detected in MNGIE patientswere reported in cultured HeLa cells after
long time culture in the presence of highlevels of thymidine in the culture medium.
These mtDNA alterations were attributed
to expanded levels of deoxythymidine
triphosphate (dTTP) and deoxyguanosine
triphosphate (dGTP) and reduced levelsof deoxycytidinetriphosphate (dCTP) anddeoxyadenosine
triphosphate(dATP).
However, no mtDNA depletion was observedin these HeLa cells (Song et al., 2003). Further
investigation revealed that this increase indTTP, under similar culture conditions,was more pronounced in non-cycling skinand lung fibroblasts leading to depletion in
mtDNA in a dThd dose and time dependent
manner (Pontarin et al., 2006). Interestingly,
mtDNA levels were recovered upon removalof the dThd from the culture medium. In orderto understand the influence of metabolites
accumulation on the creation of mtDNA
alterations, an in organelle experimental
model was used. Excess amounts of dThd
were responsible for the significant increasein mitochondrial levels of dTTP, together
leading to secondary TK2 inhibition mediatedreduction of dCTP nucleotides (GonzalezVioque et al., 2011). Subsequent studies
confirmed these findings in in vitro fibroblast
cultures and in vivo in the Tymp-/-Upp1-/mouse model and suggest that the inadequate
availability of dCTP accounts for the mtDNA
depletion observed in MNGIE (GonzalezVioque et al., 2011;Camara et al., 2014;TorresTorronteras et al., 2014).
Altogether, these studies demonstratethat indeed it is the nucleoside accumulationand subsequent reduction of dCTP
nucleotides, rather than the deficiency of
TP per se, that accounts for the molecularand phenotypic alterations in MNGIE. An
excellent illustration of this observationis the fact that TP expression in skeletal
muscles is absent, nonetheless, some but not
all MNGIE cases were reported with skeletal
muscle mtDNA deletions, histological andoxidative phosphorylation abnormalities(Papadimitriou et al., 1998;Hirano etal., 2004).
When available, although limited,
analysis of postmortem MNGIE samples isrelevant and beneficial to gain knowledge
about the molecular and pathological basisof the disease. Severe intestinal dysmotility,

225

Chapter 2

also known as chronic intestinal pseudo
obstruction (CIPO), and weight loss
are principle presentations of MNGIE.
Histopathological analysis of MNGIE
gastrointestinal samples revealed depletion in
mtDNA and mitochondrial proliferation, andconsequently cell atrophy in the muscularis
propria layer of the stomach and small
intestines (Giordano et al., 2006;Giordano
et al., 2008). Additionally, loss of interstitialcells of Cajal and morphologically abnormal
muscularis propria and ganglion cells havebeen reported (Zimmer et al., 2009). On
the other hand, the study of brain tissues of
2 MNGIE patients revealed no pathologicalproliferation of glial cells nor neuronalloss. However, the study suggested a roleof TP deficiency in impairment of blood
brain barrier, which could contribute tothe observed hyperintense T2 signals on brain
MRI scans (Szigeti et al., 2004a).
Nucleoside accumulation is detrimental
probably during the early course of

the disease, because nucleoside clearance
did not improve mtDNA content per cellor reduce COX deficient fibers after livertransplantation (De Giorgio et al., 2016).
Mitochondrial DNA instability is a hallmark
for diseases caused by defective nuclear genes
essential for mtDNA replication and repair(such as PEO1, POLG1,2) or maintenanceof dNTP pools (such as ANT1, TYMP) orothers involved in mtDNA homeostasis (suchas FBXL4) (Young and Copeland, 2016).
Mutations in PEO1, POLG and ANT1 underlie
the autosomal dominant form of progressive
external
ophthalmoplegia(adPEO),a very well characterized mtDNA disorderinvolving stalling of mtDNA replication(Van Goethem et al., 2001;Goffart et al.,2009). Therefore, stalling of Twinkle helicase
or DNA polymerase γ could be a common
pathological mechanism underlying mtDNA
instability in MNGIE, PEO and mtDNA
depletion syndrome (Hirano et al., 2001;Liu
et al., 2008).

| |Current treatments for MNGIE
In general, treatment of mitochondrial
diseases is mainly based on symptom
management and supportive care (Pfeffer
et al., 2013). Vitamin and amino acidsupplements (Tanaka et al., 1997) and exercise
therapy (Taivassalo et al., 1998) aiming
to improve mitochondrial functions arerecommended for mitochondrial myopathies.
Symptomatic management of MNGIE
consists of nutritional support (Wang et al.,2015), prevention of infections and pain relief
including interventions such as celiac plexus
neurolysis and blockage of the splenic nerve(Teitelbaum et al., 2002;Celebi et al., 2006).
Since the metabolic and mtDNA abnormalities

26

are attributed to the systemic nucleosideimbalances, clinical interventions focus on
direct removal of these metabolites to restore
the balance or by introducing the deficient
enzyme to reduce the metabolites.

Hemodialysis and peritoneal dialysis
The first hemodialysis aiming to remove
the excess amounts of nucleosides fromthe circulation was performed in 2002
(Spinazzola et al., 2002) in two MNGIE
patients followed by another in 2006. In
the first two patients, significantly reduced
thymidine levels were observed shortly
after hemodialysis, however this effect wasPathogenesis, current and prospective treatments for MNGIEtransient as thymidine levels returned to
pre-dialysis levels 3 hours after dialysis(Spinazzola et al., 2002). A progressive
reduction in Thd levels below the basal
levels was observed after repetitive dialysis
treatments in the third case (la Marca
et al., 2006). A MNGIE patient who had
peritoneal dialysis showed an improvement ingastrointestinal symptoms (such as vomiting,anorexia, abdominal pain and diarrhea)
during the continuing peritoneal dialysis forthree years with body weight gain, althoughother major symptoms including ocular andneurological abnormalities and brain MRI
signals did not change (Yavuz et al., 2007).
Another case report noted improvementof the gastrointestinal and neurologicalsymptoms, mainly the mitigation of numbness
in the hands, until nucleoside levels increased
again 15 months after continuous ambulatory
peritoneal dialysis (Ariaudo et al., 2014).

Enzyme Replacement Therapy (ERT)
Initially, platelet infusions were performed in
two MNGIE patients to restore TP enzyme
activity in the blood. This approach showed
efficient recovery of functional TP enzyme
and correction in nucleoside imbalances,
however, like dialyses, these improvements
were temporary requiring multiple treatment
sessions for long-term responses. ERT isa reliable, well-tolerated approach to replace
the deficient enzyme in a variety of lysosomal
storage disorders including Gaucher, Pompe
and Fabry disease and Sly syndrome (Wilcox
et al., 2004;Burrow et al., 2007). For MNGIE,
approaches were developed to encapsulate TP
in order to prolong the half-life of circulatory
TP enzyme and reduce the immunogenic
reactions.
These
includepolymericnanoparticles (De Vocht et al., 2009) anderythrocytes as they are permeable andaffect the plasma metabolites, such as inadenosine deaminase deficiency (Bax et al.,
2000;Moran et al., 2008). The erythrocyte
encapsulated TP (EE-TP) concept is underclinical development as an orphan ERT
for MNGIE with the first attempt carriedout in a MNGIE patient in 2008 (Moranet al., 2008). In this approach, autologous
erythrocytes were isolated from patients andloaded with recombinant E.Coli TP enzyme
in vitro via hypo osmotic dialysis. Significant
clinical improvements were observed such as
the ability to walk and climb and the recoveryof sensation and the mitigation of numbnessin hands and feet, even after 23 months aftertermination of multiple cycles of EE-TP (Baxet al., 2013). When using the EE-TP approach,there is a high risk that an immunologicalreaction is triggered against the bacterial TP,
especially if the infusions are repeated several
times, although this has not been observed(Levene et al., 2013).

2

Orthotopic liver transplantation(OLT)
Liver transplantation is a new ERT strategy
for treatment of MNGIE patients. TP protein
levels are high in healthy human liver tissues
and significantly higher than in bone marrowcells (Boschetti et al., 2014). Recently, OLT
was successfully applied in a severely affected,
25-years old MNGIE patient (De Giorgio etal., 2016). Steady state nucleoside balance wasobserved up to13 months post OLT. Slight
improvements in lower limb strength andbrain metabolism (reduced lactate levels) andstructure (reduced cerebellar mean diffusivity
values at diffusion MRI), improved quality of27

Chapter 2life scores and nutritional parameters, but not
body weight (40 kg), were observed up to six
months after OLT. When ileostomy closure was
performed, the gastrointestinal (GI) functions
and body weight declined at 13 months (37kg). Therefore, it remains uncertain whether
the mild restoration of GI function was dueto the decompressive ileostomy, instead of
the OLT. In addition, skeletal muscle mtDNA
content per cell was slightly increased after
OLT. The study suggests that the damage
in post mitotic tissues during late stages ofthe disease is irreversible despite recovery of
nucleoside balance. Therefore, biochemicalcorrection should probably be achieved prior
to irreversible damage, preferably before
the intestinal symptoms appear. Preoperative
conditioning for OLT is not required.
However, this approach requires matchedorgan donors (which are limited), involves
transplantation related risks and requires
long-term immunosuppression which all canfurther affect the quality of life of the patients.

Hematopoietic stem celltransplantation (HSCT)
Another possibility to restore TP enzyme
activity in the circulation is by HSCT. Recently,
a retrospective analysis of all HSC transplanted
MNGIE patients between 2005-2011 showedthat only nine out of 24 patients were alive up
to four years after transplantation. All ninesurvivors had normalized TP activity in theirblood while seven of them showed improved
body mass index, gastrointestinal symptoms
and peripheral neuropathy. On the other28

hand, nine MNGIE patients died mainly due
to transplant-related causes such as GVHD
and graft failure, including recipients of HLAmismatched unrelated cord blood transplants,
while the remaining six patients died of diseaseprogression. The recommendations of thisstudy included transplantation of a sufficientnumber of cells, because in some patientsthe graft was rejected, and to consider moreclosely HLA matched donor cells, becauseof the large number of GVHD observed inthis retrospective study, and to transplantat an earlier age before major organ damage
has occurred (Halter et al., 2011;Halter etal., 2015).
The poor physical state of MNGIE patients
when they enroll HSCT trials increases the risk
for transplantation related complications
caused by conditioning regimens andimmune-suppressants. Other problems may
arise from the drugs used in HSCT which arepotentially harmful to mitochondria such as
cyclophosphamide (Mariana Ponte Cardoso
et al., 2015). Therefore, MNGIE patients aretreated with Busulfan and fludarabine priorto HSCT, following the recommendationsof MNGIE consensus meeting in 2011
(Halter et al., 2011). For MNGIE patients
who develop liver cirrhosis, AHSCT should
be contraindicated and OLT would be
the treatment of choice (Finkenstedt et al.,2013). Pre-existing liver cirrhosis complicates
liver failure which may develop after AHSCT
due to multiple factors such as viral infectionsin immunocompromised recipients or due tohepatotoxic conditioning drugs.

Pathogenesis, current and prospective treatments for MNGIE| |Pre-clinical experimental approaches
towards therapy
Models of MNGIEIn organello experiments and human MNGIE
fibroblasts were used for highlighting partsof the molecular mechanism of MNGIE(Gonzalez-Vioque et al., 2011;Camara et al.,2014). To study therapeutic interventions,such as gene therapy, in vivo models are
required. A mouse model was developed bytargeted disruption of exon 4 of the TYMP
gene to generate Tp-/- mice. In contrast tohuman TP, murine TP degrades both dThd
and dUrd; Tp-/- mice were crossed with
Upp1-/- to generate the Tymp-/-Upp1-/- mice,
which are currently the only relevant in vivo
animal model (Haraguchi et al., 2002;Lopezet al., 2009). Tymp-/-Upp1-/- mice show
increased levels of the purine nucleosides
dThd and dUrd in plasma and tissues.
Diffuse leukoencephalopathy manifests lateduring the lifetime of these animals, aroundthe age of 22 months (Lopez et al., 2009).
Other symptoms associated with MNGIE,
such as decreased motor coordination andgastrointestinal features have not beenreported in this mouse model. Brain mtDNA
depletion was not consistently found in thismouse model (Lopez et al., 2009;TorresTorronteras et al., 2011;Camara et al.,2014). Therefore, high doses of exogenous
nucleosides were administered to exacerbate
the mitochondrial phenotype (Garcia-Diaz etal., 2014), an approach that was rationalized
by the lower nucleoside levels in Tymp-/-Upp1-/mice compared to MNGIE patients. Mice wereon an exogenous dThd and dUrd diet for a longtime (24 months) before pronounced mtDNA
depletion, diffuse leukoencephalopathy andmotor abnormalities were observed.

Experimental approaches have been
explored for treatment of MNGIE; among
which experiments performed by Camara etal which suggest that modulation of dNTP
metabolism through increasing the availabilityof dCTP or inhibition of its catabolism canindeed reverse and prevent, at least, dCtd
imbalance. A strategy that can be applied forother similar mitochondrial disorders thatare caused by altered nucleosides and dNTP
metabolism, for example in disorders caused
by mutations in TK2 or DGUOK deficiency(Camara et al., 2014).

2

Gene Therapy
The Tymp-/-Upp1-/- mouse model has also beenused for testing potential curative treatments.
A recently investigated strategy is the use ofgene therapy. Both lentiviral (LV) and adenoassociated viral (AAV) vector mediated
TYMP gene transfer have been evaluated in
pre-clinical studies for treatment of MNGIE.

AAV-mediated liver directed genetherapy
AAV vector gene therapy has been exploredin clinical trials for a variety of inherited
and acquired diseases (Naldini, 2015).
The main limitation of this approach is
the human immune response to AAV capsid,
as demonstrated in hemophilia B trials. In
one of the first AAV trials targeting the liver,therapeutic levels of coagulation factor IX(FIX) were achieved at a high vector dose
(2×1012 vector genomes per kilogram ofbody weight, vg/kg). Nonetheless, this highvector dose was associated with an early
decline of FIX (~8 weeks after treatment)
29

Chapter 2

due to T-cell immunity against AAV capsid
antigens eliminating transduced hepatocytes(Manno et al., 2006).
The hybrid vector AAV2/8 with modified
molecular configuration (packaged double
stranded genome) and improved cassette
design (codon optimized hFIX) to enhancetransduction and translational efficiency was
explored in a hemophilia B trial (Nathwani etal., 2006). Stable FIX expression diminished useof the costly FIX concentrate and importantly,
clinical improvement was achieved ina dose dependent manner. The least bleeding
episodes were seen in recipients of the highest
AAV dose (steady state 5% of normal levels
at a single vector peripheral vein infusion of
2×1012 vg/kg up to four years) (Nathwani etal., 2014). For MNGIE, an AAV2/8 expressing
human TYMP under the control of hepatic
promoter was used for treatment of Tymp-/Upp1-/- mice (Torres-Torronteras et al., 2014).
Low AAV doses (2×1011 vg/kg ) were sufficient
to reduce nucleoside imbalances to normal
levels in liver, skeletal muscle and brain for up
to eight months, while higher doses reduced
nucleosides below detection levels. However,
only at higher doses (>2×1011vg/kg) TP
activity was increased in the liver (but not inskeletal muscle or brain).
In light of the clinical data of the hemophilia
B trial which shows that clinical improvement
was AAV dose dependent (Nathwani et al.,2014), the question for MNGIE is whether
or not the low AAV dose would be sufficient
to reverse a clinical phenotype beyond
biochemical correction. MNGIE mousestudies failed to report any relevant clinicalphenotype in Tymp-/-Upp1-/- mice, andtherefore the potential or required dosage tocure it has not been demonstrated (TorresTorronteras et al., 2011;Torres-Torronteras et30al., 2014). Importantly, upon AAV treatment
nucleosides accumulation was not reduced in
the intestine of treated mice at the highest dose
(1013 vg/kg) administered. Since the intestines
are heavily affected in MNGIE patients it isimportant to obtain evidence of correction inthis organ. Preclinical studies in hemophilicdogs and non-human primates could predict
the therapeutic dose in human trials (Manno
et al., 2006;Nathwani et al., 2014) if thatis the case for MNGIE too, biochemicalcorrection in the intestine might require
improved expression cassettes to enhanceprotein production, targeting the expression
to major affected organs, or the less favorable
option of using higher AAV doses (>1013 vg/
kg). High doses, for example 7.2 × 1012 vg/
mouse were sufficient to transduce 100%of mouse hepatocytes (Nakai et al., 2005).
Such high doses might be required for genetherapy of systemic diseases, i.e. when nonhepatic tissues are also affected, as in MNGIE.
However, these high AAV doses are likely tocause hepatocellular toxicity, biodistribution
to other unwanted organs and shedding of
the AAV, enhanced risk of eliciting immunity
towards the viral capsid and increased costsof virus production. Immunity against
ectopic TP might be an additional concern
for MNGIE patients, therefore prophylactic
immunosuppression

might

be

required.

Additional pre-clinical studies have to address
the possibility of an immune response against
ectopic TP in previously untreated patients.
In addition to increased liver TP activity
correlating with vector dose, an unexpected
increase in liver dGTP of Tymp-/-Upp1-/- mice
was observed in a dose depend manner as well,although the consequences of this increase
are unknown. Together these findings suggest

Pathogenesis, current and prospective treatments for MNGIE

that studies into optimal dosing of AAV may
be required for clinical application.
Human AAV trials should be carried out
cautiously as they can reveal complications
that were not observed during preclinicalstudies. An example is the early decline in
FIX expression (Manno et al., 2006) andhepatotoxicity observed in 4/6 recipients ofa high AAV dose (2×1012 vg/kg) (Nathwani
et al., 2014), due to immunity against AAV
capsid. MNGIE patients are often > 12 years
and probably have been pre-exposed to AAV
and consequently can mount strong immune
responses to AAV. Therefore, individuals
with neutralizing antibodies to AAV should
be excluded from clinical trials to avoid an
immune response towards AAV. Anotherconcern is the durability of transgene
expression considering the longer lifespanof humans, compared with the animals in
preclinical studies, and the potential need forrecurrent AAV injections, especially at lower
vector doses. In this respect hematopoietic
stem cell gene therapy (HSCGT) would
provide a preferable option as a single, long
lasting intervention method. Additional
concerns related to AAV mediated genetherapy include purity of AAV preparations
and manufacturing costs (Mingozzi andHigh, 2013).

LV-mediated hematopoietic stem cellgene therapy (HSCGT)The encouraging therapeutic outcomes andfavorable safety profile renders LV-HSCGT
an attractive therapeutic approach fora variety of hereditary metabolic disorders(Wagemaker, 2014), and is potentiallyadvantageous over AHSCT for certain
selected diseases (Naldini, 2015). Proof ofconcept of HSC gene therapy was obtainedin Tymp-/-Upp1-/- mice (Torres-Torronteras
et al., 2011) using a phosphoglycerate kinase
promoter driving native human TYMP
cDNA and a GFP reporter in hematopoietic
cells resulting in biochemical correctionin peripheral blood (Torres-Torronteras
et al., 2011). More recently, we developed
clinically applicable LVs that carry human
TYMP cDNA, and demonstrated long-term
biochemical correction in Tymp-/-Upp1-/mice at low vector copy number (VCN). Ourdata demonstrates the feasibility to further
develop clinical protocols for HSCGT for
MNGIE (Yadak et al., 2015). Similar results ina long-term follow up of 20 months confirms
the correction of biochemical imbalances
which was maintained at low vector copy
number and chimerism (Torres-Torronteras
et al., 2016).
In HSCGT for MNGIE, HSCs are isolated
from MNGIE patients, transduced ex vivo
by LV vectors carrying a functional copy
of TYMP and infused back into the patient(Figure 2A). The newly formed HSCs andits progenitors produce TP which catabolize
the excess amounts of nucleosides (Figure2B). Since the patient’s own stem cells areused, GVHD is not a concern. However, mild
prophylactic immunosuppression maybe
required to prevent possible immune reaction
against the TP transgene.
Myeloablative pre-conditioning might
be necessary for high levels of engraftment
in other metabolic disorders, such as
metachromatic leukodystrophy (MLD), due
to lack of selective advantage of gene modified
cells. In particular, busulfan myeloablative
conditioning is used in MLD patients for
depletion of endogenous microglia andmobility of gene modified monocytes throughthe blood brain barrier (BBB) (Capotondo et231Chapter 2al., 2012). The contribution of gene modified
microglia to correct biochemical imbalances
has never been explored in MNGIE. However,murine gene therapy studies using LV andAAV vectors implicate reversal of nucleosideimbalance at low or possibly no increasein brain TP activity (Torres-Torronteras etal., 2014;Torres-Torronteras et al., 2016). In
liver directed AAV2/8 gene therapy, it is notexpected that brain cells will be transduced.
In HSCGT, gene modified monocytes are
expected to migrate to brain and differentiate
into microglia. Nonetheless, the results ofthe HSCGT MNGIE mouse study do notrule out the potential that gene modified
microglia can contribute to correctionof brain biochemistry and phenotype,although this might not be necessary if
ectopic expression outside the brain is highenough. When transduction efficiency
is high enough, significant TP activity
can be measured in the brain, indicatingthat transduced microglia might residein the brain after long-term follow-up. To
that end, two potential mechanisms mightact synergistically to normalize the brain
nucleoside levels, a systemic ectopic source
and one local contribution of gene-modified
cells (Figure 2B).
Potential options and future research
for application in MNGIE patients include
alternative conditioning strategies to obviate
the cytotoxicity related to myeloablative
conditioning and strategies to enhance
the quality of infused gene modified HSCs.
One approach is to mobilize endogenous
HSCs into peripheral blood in order tocreate (space) in the bone marrow for
the infused donor HSCs to engraft (Chenet al., 2006). Human granulocyte colony
stimulating factor (G-CSF) was sufficient in
32

immunocompromised mice (Huston et al.,2014), probably due to the selective advantageof the gene modified cells, however more
stringent agents might be required in normal
immunocompetent mice. A possibility is
G-CSF in combination with the more potent
HSCs mobilizer plerixafor (a specific CXCR4
antagonist) or the selectins inhibitor fucoidan.
Such regimens probably require additional
mild chemotherapeutics, in particular if
the gene corrected TP-expressing HSCs lackselective growth advantage to overcome
host cells. These HSCs mobilizers act via
different mechanisms, therefore parameterssuch as the optimal dose and time frame fortransplantation after mobilization need to beestablished in relevant pre-clinical models.
Alternatively, targeting specific endogenous
hematopoietic populations might reduce
the off-target toxicity related to the common
non-specific conditioning (Aiuti and Naldini,2016). Examples include inhibiting c-kit,
a HSC tyrosine kinase cell surface antigen(Xue et al., 2010) and the recently developed
immunotoxin against hematopoietic stem
cells (CD45-SAP) (Palchaudhuri et al., 2016).
Strategies such as ex vivo expansion ofgene modified HSCs can improve the qualityof the infused gene modified cells and enhancethe outcome of gene therapy (Watts et al.,2011). In particular when combined withadditional approaches to enrich for HSCs,
preserve stemness of- and enhance homing
and engraftment ability of gene modified
HSCs (Psatha et al., 2016). Ultimately, thisapproach combined with improved mild preconditioning protocols, could benefit patientsin poor health condition at transplantation,such as in MNGIE patients.
A risk of HSC gene therapy is insertionalmutagenesis. The first HSC gene therapy

Pathogenesis, current and prospective treatments for MNGIE

trials used gammaretrovirus (γ-RV) based
vectors for treatment of X-linked severe
combined immunodeficiency (SCID-X1)(Gaspar et al., 2004;Hacein-Bey-Abina et al.,2010), adenosine deaminase (ADA-SCID)(Aiuti et al., 2002), chronic granulomatous
disease (CGD) (Ott et al., 2006) andWiskott-Aldrich syndrome (WAS) (Boztug
et al., 2010). Although efficient correctionof immunodeficiency was achieved in mostpatients in SCID-X1, CGD and WAS trials,
lympho-proliferative disorders (HaceinBey-Abina et al., 2003a;Hacein-Bey-Abinaet al., 2003b;Hacein-Bey-Abina et al.,2008;Howe et al., 2008;Braun et al., 2014) andmyelodysplasia (Stein et al., 2010) developed
secondary to γ-RV vector integrations withinor nearby proto-oncogenes. In addition to
the preferred integration profiles over γ-RV
vector (Deichmann et al., 2007;Gabriel etal., 2012;Cattoglio et al., 2007), LV efficiently
transduce non-cycling primitive HSCs andunder minimum culture conditions (Naldini
et al., 1996;Guenechea et al., 2000). Therefore,
attention was focused on development of
LV as a relatively safer approach, leading
eventually to development of thirdgeneration self-inactivating HIV derivedvectors (SIN-LV) (Dull et al., 1998;Zufferey
et al., 1998). Several pre-clinical studies
indicate the reduced genotoxicity of SIN-LV
vectors compared with γ-retroviral vectors,
in particular SIN-LV common integrationsites (CIS) revealed no preference ofintegration near proto-oncogenes (Montini
et al., 2006;Modlich et al., 2009;Romero etal., 2013;Zhou et al., 2013;Biffi et al., 2011).
Since then, SIN-LV vectors have been applied
successfully in ongoing clinical trials fora variety of metabolic (Cartier et al., 2009;Biffi
et al., 2013) and immunodeficiency disorders(Aiuti et al., 2013), and no adverse events have
yet been reported in these trials. Moreover,
the therapeutic benefits without toxicity
related to transgene expression and biosafetyof SIN-LV vectors has been further validated

2

through a growing body of recent preclinicalstudies supporting the initiation of clinical
trials, for example for β-thalassemia (Negre
et al., 2015) and mucopolysaccharidosis I
disease (Visigalli et al., 2016).
Furthermore, selective advantage for
growth and differentiation conferred by
the therapeutic transgene expression increases
the potential risk for proliferative disorders,
this was reported in some immunodeficiency
conditions (Aiuti and Roncarolo, 2009). For
metabolic disorders, for instance lysosomal
storage disorders , however, most studies showthat enzyme positive cells have no selective
advantage (Bernardo and Aiuti, 2016), which
is most likely the case in MNGIE as well. To
improve safety, technologies such as ex vivo
expansion of gene modified HSCs may permit
for safety assessment (to some degree) prior totransplantation, by analysis of LV integrationsites (Watts et al., 2011) (Figure 2A).

AAV mediated GT or HSCGT?
Regardless of the type of viral vector used
for gene therapy, the chosen strategy should
provide long-term expression of the gene ofinterest without side effects in the host. It is
important to apply a well-defined vector dosethat is sufficient to reverse the biochemical
andnucleotide

imbalance

withoutany

potential side effects. In particular, abnormal
overexpression of the TP enzyme is detected indifferent tumor types, including non-small celllung-, colorectal-, breast-, gastrointestinal-,
and hepatic cancers (Koukourakis et al.,33

Chapter 2

1997;Mori et al., 2000;Ikeguchi et al.,
2001;Nakayama et al., 2005;Mitselou et al.,2012) and correlates with a worse prognosis
in colorectal cancer patients (Takebayashi
et al., 1996). Besides, disturbance of dNTP
pools can be a trigger for cell cycle arrest
and apoptosis (Oliver et al., 1996) (Kumar etal., 2010).

The medical condition of the patient canalso influence the choice of the vector systemfor clinical application. For terminally illpatients, the AAV approach could be mostsuitable to avoid the risks associated withthe pre-conditioning for transplantation in
autologous HSC gene therapy or if a suitable
HSCs donor for AHSCT is lacking.

| |Concluding remarks
The lack of mitochondrial histone protection,
the limited repair capacity and oxidized
dNTPs contributing to mismatch errors(Alexeyev et al., 2013) all make mitochondria
more susceptible than nuclear DNA tomutagenesis. It has become evident that itis the systemic accumulation of nucleosides
in MNGIE (Di Meo et al., 2015) that causes
imbalances in mitochondrial dNTP pools.
However, the mechanism by which it
causes mtDNA alterations is still unknown.
Although the current treatments focus on
restoration of TP enzyme activity and/ or
elimination of accumulating metabolites,
further understanding of cellular mechanisms
involved in maintenance of mtDNA integrity
and copy number can provide targets forclinical intervention for MNGIE and possibly
other mitochondrial disorders.
Platelet infusions, hemato/peritoneal
dialysis and erythrocyte encapsulated TP
enzyme replacement therapy could be used
to provide biochemical correction. AAV genetherapy and lentiviral HSCGT are potential
curative options as evidenced by the promisingpre-clinical results in Tymp-/-Upp1-/- mice
. OLT is a promising emerging treatment
and should currently be the treatment of

34

choice for MNGIE patients with pre-existing
liver failure. Allogeneic HSCT has risks
of graft failure, GVHD and conditioningrelated toxicity. Milder conditioning may be
applicable in HSCGT, and treatment should
preferably be applied at an early age. Novel
strategies are being explored to improve
the safety and efficiency of viral based genetherapy, ultimately for MNGIE patients aswell. These include strategies to enhancetransduction, improve engraftment of genemodified HSCs and limit transplantation
related toxicity, and others to overcome
the limitation of AAV capsid triggered
immunity by means of novel serotypes andimproved transcription cassettes.
MNGIE patients should receive suitable
treatment promptly before permanent
damage occurs, which can be challenging,as MNGIE patients are often diagnosed lateduring disease progression in a poor healthcondition. Because TP activity and nucleosidelevels can be routinely measured in blood
samples, MNGIE should be considered to be
included in newborn screening programs,
similar to other (neuro) metabolic disorders
for early diagnosis and treatment (Carlson,
2004;McHugh et al., 2011).

Pathogenesis, current and prospective treatments for MNGIE| |List of abbreviations
AAV: adeno-associated virus; ADA-SCID:
adenosine
deaminase-severe
combined
immunodeficiency;
AHSCT:
allogeneic
hematopoietic stem cell transplantation;
ANT-1: adenine nucleotide translocase
type 1; BBB: blood brain barrier;
CGD: chronic granulomatous disease; CIPO:
chronic intestinal pseudo obstruction; CIS:
common integration site; CNT: concentrative
nucleoside transporter; CPEO: chronic
progressive
external
ophthalmoplegia;
dATP:
deoxyadenosine
triphosphate;dCTP:deoxycytidinetriphosphate;dGTP:deoxyguanosine
triphosphate;
DGUOK: deoxyguanosine kinase; dTTP:
deoxythymidine
triphosphate;dNTP:
deoxyribonucleoside triphosphates; dThd:
thymidine;dUrd: deoxyuridine; ECGF1:
platelet-derived endothelial cell growth
factor; EE-TP: erythrocyte encapsulated TP;
ENT: equilibrative nucleosides transport;
ERT: enzyme replacement therapy; FBXL4:

F-box and leucine-rich repeat protein
4; FIX: coagulation factor IX; G-CSF:
granulocyte colony stimulating factor; γ-RV:
gammaretrovirus; GVHD: graft versus hostdisease; HSCT: hematopoietic stem celltransplantation; HSCGT: hematopoietic
stem cell gene therapy; LHON: Leber’s
hereditary optic neuropathy; LV: lentivirus;
MDS: mitochondrial DNA depletion
syndrome;
MNGIE:
mitochondrial
neurogastrointestinal encephalomyopathy;
MELAS: mitochondrial encephalomyopathy,
lactic acidosis, and stroke-like episodes; NT:nucleoside transporter; OLT: orthotopic livertransplantation; PEO: progressive external
ophthalmoplegia; POLG: DNA polymerase
subunit gamma; RRM2B: Ribonucleotide
Reductase M2 B; SCID- X1: X-linked severe
combined immunodeficiency; SIN-LV: selfinactivating LV; TK2: thymidine kinase 2;
TYMP: thymidine phosphorylase; WAS:
Wiskott-Aldrich Syndrome.

2| |Conflict of interest statement
The authors declare no conflict of interest.

| |Author contributions
RY framed the structure of the review, analyzed
the literature and wrote the manuscript; PS,
MW participated in the literature analysis;
NP, IF participated in the literature analysis
and supervised the writing.

All authors discussed the topic and provided
intellectual feedback to the article.
All the authors read and approved
the manuscript.

| |Funding
We would like to acknowledge the financial
support of Join4energy, the Sophia Foundation(SSW0645) and the Stichting NeMo.

35

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47

Development of a highly efficientlentiviral gene transfer protocol forhematopoietic stem cells
Niek P. van Til*,1,2 Rana Yadak*,1 Valentina Buffa,1 Marshall W. Huston,1
Fatima Aerts-Kaya,3 Yvette van Helsdingen,1 Trudi P. Visser,1
Monique M. Verstegen,1 Chiara Bovolenta,4 Constanze Radek,5
Ian C. D. Johnston,5 and Gerard Wagemaker1,3,6
1
Department of Hematology, Erasmus University Medical Center, Rotterdam, The Netherlands;
Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht, The Netherlands;
3
Center for Stem Cell Research and Development, Graduate School of Health Sciences, Hacettepe
University, Ankara, Turkey; 4MolMed S.p.A., Milan, Italy; 5Miltenyi Biotec GmbH, Bergisch Gladbach,
Germany; 6Raisa Gorbacheva Memorial Research Institute for Pediatric Oncology and Hematology,
Saint Petersburg, Russia; * Authors contributed equally.

2(To be submitted)

3Chapter 3| |Abstract
Efficient ex vivo hematopoietic stem/progenitor cell (HSPC) gene transfer protocols requiring
low vector doses would reduce the number of vector particles required per patient and minimize
the risk of genotoxicity by keeping vector copy number (VCN) low. With current lentiviral (LV)
protocols, a high proportion of gene-modified HSPC requires a high vector dose, which often
results in an undesirably high number of transgene copies per cell. To reduce genotoxicity risks
and to control transgene expression levels, a single integration per cell is preferred. We observedthat overnight transduction of VSV-G packaged LV expressing EGFP of lineage-depleted (Lin-)
mouse bone marrow cells (BM), rhesus CD34+ BM and human CD34+ derived progenitor
cells is markedly improved by solely increasing the cell density with a proportional increaseof transducing units (TU)/mL. The principle also partly abrogates the restriction of rhesus
monkey CD34+ cells for HIV-1 vectors. Additionally, we show that overnight transduction withthe cytokine TPO is sufficient for efficient transduction of mouse and human HSPC progenitors,
while maintaining stem cell properties in vivo.
We conclude that highly efficient and short LV HSPC transduction protocols with single
cytokine additions results in an optimal number of integrations per cell. It is of note thatthe method will drastically reduce volumes of virus stocks and transduction reagents required
for ex vivo LV-mediated gene-transfer.

50

Critical parameters for efficient HSPCs gene transfer| |Introduction
Gammaretroviral vector (gRV) hematopoietic
stem/progenitor cell (HSPC) gene therapy
has been demonstrated to be successful
for long-term treatment of X-linked severe
combined immunodeficiency (SCID-X1)1,
2
and adenosine deaminase (ADA)-SCID.3
Unfortunately, gRV have a risk for insertional
oncogenesis and consequently, safer selfinactivating (SIN) RVs with reduced-risk
genotoxicity have been developed.4 Third
generation SIN lentiviral vectors (LV)5, 6 may
further decrease genotoxicity risks since they
feature a reduced likelihood of upregulating
proto-oncogenes due to reduced affinity
to integrate near transcription start sites
compared to RV. 7 The use of eukaryotic
promoters, whether constitutively active
or cell type-specific, rather than viralpromoters could further improve the vector
safety profile.
Furthermore, LVs are more efficient intransducing primary human cells than gRVs.
As LV vectors require less transduction
time and have no prerequisite for growth
stimulation or cell division to integrate into
the genome the efficiency is enhanced byaddition of cytokine cocktails that stimulate
cell cycling.8 Some recent HSPC-based
clinical gene therapy trials have thereforechanged to lentiviral vectors, e.g. for X-linked
β-thalassemia,10
adrenoleukodystrophy,9andmetachromatic
leukodystrophy11
Wiskott-Aldrich syndrome.12
In most ex vivo LV clinical trials, likethe gRV protocols, HSPCs are currently
pre-stimulated with a cytokine cocktail that
includes interleukin 3 (IL-3), even though thiscondition results in the partial loss/reductionof their stemness.13 Furthermore, HSPCs are

incubated for multiple rounds with a highvector dose and transduced in dishes precoated with the CH296 human fragment offibronectin (RetroNectin) and/or protamine
sulfate 9-12, 14 to reduce the distance between
the vector and the target cells.
Previous observations with gRV15 havedemonstrated that one of the major limiting
factors in viral transduction is the distance
between the virus particle and the target
cell. This could be reduced by increasing
either the cell density or the viral vector
concentration, or both.
We examined whether improvement of
the physical LV transduction parametersfor HSPCs combined with a minimal
stimulation protocol, could lead to a more
effective generation of genetically modified
HSPCs with intact repopulation andhoming potential.
To address this, feasibility of increasedmouse, rhesus and human HSPC densities
and LV multiplicity of infection (MOI) weretitrated in vitro to obtain optimal transduction
efficiencies. For mouse HSPCs, the long term
repopulation capacity of the transduced
cells at varying MOIs was tested in vivo inprimary and secondary recipients, including
GLP grade LV vector. Furthermore, to obtain
minimal cytokine combinations required for
efficient transduction of mouse and human
HPSCs, while pertaining in vitro progenitors
and in vivo repopulation capacities. In
particular, SCF, TPO, Flt3-L combinations
were tested, which are cytokines commonlyused in clinical trials,11, 12 with homemadeor commercially improved culture media.
This enhanced gene transfer protocol would
contribute to reduced risks of genotoxicity,

351

Chapter 3

by using low MOI, and reduced transduction
time, by increasing the cell density andtheoptimalcytokine
combinations.
Moreover, minimally used cytokine cocktails
permitting sufficient HSPC proliferation/differentiation at low vector copy number,would add commercial value by significantly
increasing the number of patients treated per
LV batch and reduce the amount of cytokine
for transduction protocols.

| |Materials and Methods
Animals
Normal BALB/c mice were obtained fromour own breeding colony, CD45.1 andCD45.2 mice from Jackson Laboratory(Maine, USA). Purpose bred rhesus monkeys(Macaca mulatta) were free of intestinal
parasites, and seronegative for herpes B,
simian T-lymphotropic viruses, simian
immunodeficiency virus, Ebola and Hepatitis
B viruses. All animal experiments werereviewed and approved by an ethical committeeof Erasmus MC, Rotterdam in accordancewith legislation in The Netherlands.

Lentiviral vector production
The third generation self-inactivating LV
vectors pRRL.PPT.SF.EGFP.bPRE4*.SIN (SF.
GFP) containing the spleen focus forming
virus (SF) or the phosphoglycerate kinase(PGK) promoter driving EGFP have been
described previously.5, 6, 16, 17
The lentiviral vectors were produced bystandard calcium phosphate transfection of
HEK 293T cells with the plasmids pMDL-g/pRRE, pMD2-VSVg and pRSV-Rev.5, 6
Lentiviral vectors were concentrated byultracentrifugation for 2 hours at 20,000
r.p.m. at 4°C and stored at -80°C. Titers
were determined by serial dilution on HeLa
cells and GFP expression was measured byflow cytometry five days post-transduction.18
The good laboratory practice (GLP) lentiviral
vector lots were prepared by MolMed S.p.A.
52(Milan, Italy) as previously described11, 12
with the potency/composition mentioned insupplementary table S2.

Hematopoietic stem cell enrichment
and lentiviral transduction
Donor bone marrow was extracted from both
femurs and tibias of eight to twelve week-old
male mice and hematopoietic progenitors
were purified by lineage depletion (Lin-)
according to the manufacturer’s protocol (BDBiosciences, USA). After enrichment, Lincells were transduced by the GFP SIN-LV
overnight at different cell densities or different
multiplicity of infections (MOIs), as indicated,either in serum free modified Dulbecco’s
medium with supplements (Standard Lab
Medium, SLM).19 Alternatively, StemMACS
HSC Expansion Media XF, human (Miltenyi
Biotec, Bergisch Gladbach, Germany) andcombinations of growth factors, i.e. murine
stem cell factor (mSCF) 100 ng/mL, human
FMS-like tyrosine kinase 3-ligand (hFlt3-L)
50 ng/mL and murine thrombopoietin(mTPO) 20 ng/mL (STF) was used.16 After 18
hours of incubation at 37°C, under normoxia,
the cells were washed and expanded in
the same culture medium supplemented
with the growth factors mentioned above for7 days under hypoxia conditions (3% O2),
for further assessment of GFP expression,
VCN and colony forming assays. For in vivo
experiments, after the overnight transduction,

Critical parameters for efficient HSPCs gene transfer

a titration of transduced BALB/c or CD45.1
Lin- cells were injected in the tail vein of 6
Gy sub-lethally irradiated eight to twelve
week-old female BALB/c or CD45.2 recipients
respectively (1.4×105 to 3×106 Lin- cells).
For rhesus CD34+ cells, bone marrow(BM) aspirates were isolated as previously
described.19 After cell separation, BM
suspension was frozen in liquid nitrogen.
After thawing, the BM cells were washed with
Hank’s with HEPES, and CD34+ cells were
subsequently isolated by immunomagnetic
separation using an IgG2a antibody against
CD34 (mAb 561 kindly provided by T.
Egeland, University of Oslo, Oslo, Norway),
which was non covalently linked to rat-antimouse IgG2a beads (Dynal, Oslo, Norway).
CD34+ cells devoid of the anti-CD34 antibody
were recovered using a polyclonal antibody
against the Fab part of the anti-CD34 antibody(Detachabead, Dynal). Recombinant fulllength rhesus monkey TPO produced by
Chinese hamster ovary cells was supplied by
Genentech Inc. (South San Francisco, CA),19 and added to the same concentrations of
hSCF and hFlt-3L as mentioned above.
For human umbilical cord blood(UCB) CD34+ cells, UCB samples wereobtained from umbilical cords of full-term
normal pregnancies after informed consent
in conformity with legal regulations in
The Netherlands. Mononucleated cells
were isolated by Ficoll density gradientcentrifugation (1,077 g/cm2; NycomedPharma AS, Oslo, Norway), and were
cryopreserved in 10% dimethylsulphoxide(DMSO), 20% heat-inactivated fetal calf
serum (FCS), and 70% Hanks’ Balanced
Salt Solution (HBSS; GIBCO, Breda,
The Netherlands). After thawing by stepwise
dilution in HBSS containing 2% FCS andDNAse, the cells were washed with HBSS
containing 1% FCS and CD34+ cells were
purified from mononuclear cells by using
the CD34+ MicroBead Kit, human (Miltenyi
Biotec)accordingtomanufacturer’s
instructions, and used for gene transduction
experiments. Overnight transductions were
performed in SLM, or StemMACS HSC
expansion media XF (Miltenyi Biotec) withhSCF 100 ng/mL, hFlt3-L 50 ng/mL andhTPO 20 ng/mL (STF) or 20 ng/mL TPO
alone under normoxia conditions. After 18
hours of incubation at 37°C, the cells were
washed and expanded in the same culture
medium supplemented with human SCF
50 ng/mL, human TPO 20 ng/mL, human
FGF-1 10 ng/mL, mouse IGF-2 20 ng/mL,
human angiopoietin like protein-3 200 ng/
mL and heparin 10 μg/mL (STIF-A3) for 7
days under hypoxia conditions (3% O2) for
further assessment of GFP expression, VCN
and colony forming assays.
Buffy coats or apheresis harvests
from healthy donors were obtained fromthe University hospitals in Dortmund
and Cologne. Bone marrow samples werepurchased from AllCells (Alameda, USA).
These studies were performed according toestablished ethical guidelines, and all blood
donors gave informed consent. Peripheral
blood mononuclear cells (PBMC) wereseparated by using Ficoll–Hypaque (GEHealthcare, Bio-Science) density gradientcentrifugation. PBMC obtained frombuffy coats were pooled before magnetic
enrichment of adult CD34+ cells using
the CD34 MicroBead Kit, human (Miltenyi
Biotec)accordingtomanufacturer’s
instructions. CD34+ cells were obtained fromapheresis harvests or bone marrow samples
using CD34 Reagent and the CliniMACS®plus

3

53

Chapter 3

or CliniMACS® Prodigy instrument according
to manufacturer’s instructions. CD34 cells+

isolated from buffy coats were immediately
taken into culture for 24 hours in 24-well

Immunological phenotyping

plates at 4×10 cells per mL in StemSpan

Murine peripheral blood was collected
monthly by retro-orbital puncture underisoflurane anesthesia in EDTA tubes. For
FACS analysis, GFP expression in erythrocytes
and platelets was measured in peripheral
blood suspended in 1 mL 0.6% NaCl buffer.
Separately, erythrocytes were removed withlysis buffer (0.15M NH4Cl, 0.1M EDTA) andleukocytes were washed two times with Hank‘sbalanced salt solution (HBSS, Invitrogen)containing 0.5% bovine serum albumin (BSA)
and 0.05% sodium azide (HBN). Leukocytes
were incubated for 30 minutes at 4°C in
HBN containing 2% heat-inactivated normalmouse serum and antibodies against CD45.1,
CD3, CD19, CD11b, directly conjugatedrespectively to APC-Cy7, APC, PE, PerCPCy5.5 (BioLegend, London, UK). Leukocytes
were resuspended in 40 µL HBN and measured
for GFP expression by flow cytometry.

5

SFEM medium (Stem Cell Technologies,
Grenoble,

France)supplemented

with

Flt-3L, SCF, IL-3 and IL-6 (StemSpan CC100
Cocktail, Stem Cell Technologies). After
24-hour pre-activation, the concentration of
the CD34+ cells was adjusted to 105, 106 or 107
cells per mL and the cells were transducedwith VSV-G pseudotyped 3rd generation LVs
encoding a GFP transgene (pRRL.ppt.PGK.
GFP.wpre.sin-18), that had been concentrated
by low-speed centrifugation, at an MOI of 10
or 100. CD34+ cells isolated from apheresis
samples or bone marrow were cultivatedin CellGro SCGM medium (Cellgenix,
Freiburg, Germany) or StemMACS HSC
Expansion Media XF, human (Miltenyi
Biotec) supplemented with Flt-3L 300 ng/mL,
SCF 300 ng/mL, IL-3 60 ng/mL and TPO 100
ng/mL (Miltenyi Biotec). After 24-hour preactivation, the concentration of the CD34+
cells was adjusted to 105 – 107 cells per mL andColony-forming assay

the cells were transduced with clinical grade

Semisolid methylcellulose 80% of the total
volume (MethoCult M3231,and MethoCult
H4230 respectively, Stem cell technologies,
France) was seeded with fresh or overnight
cultured mouse Lin- cells (2x103 /ml) or UCB
CD34+ cells (1x103 /ml) or an equivalentof 500 culture expanded cells suspended in
10% of the total volume. Total volume wascompleted with IMDM supplemented withmurine SCF 100 ng/mL, murine IL-3 30
ng/mL and murine GM-CSF 30 ng/mL for
murine colony forming unit- granulocyte
monocyte (CFU-GM) or hu-SCF 100 ng/
mL, hu-IL-3 30 ng/mL, hu-GM-CSF 20 ng/

LV containing a GFP expression cassette(details supplementary table S2) at an MOIof 10, 20 or 100. Cells transduced at 107 permL were diluted to 106 per mL 24 hours posttransduction. Transduction efficiencies werecompared to a standard transduction protocol
11, where pre-activated cells were seeded at 106

cells per mL on RetroNectin-coated plates
before 2 rounds of transduction for 24 hours
at an MOI=100. Cells were rested for 12 hours
between the two transduction rounds. Cells
were assayed for GFP expression between
5 and 14 days post transduction by flow
54

cytometry using a MACSQuant® Analyzer(Miltenyi Biotec).

Critical parameters for efficient HSPCs gene transfer

mL and hu-EPO 4U/mL for human CFU-GM
and burst forming unit- erythroid (BFU-E).
Duplicate cultures were incubated at 37 °C
in a humidified atmosphere containing 5%
CO2 and colonies were scored 7 or 14 days formouse and human colonies respectively.

Quantitative PCR of lentiviral
integrations
The lentiviral copy number per cell (VCN)
following murine engraftment was determinedon genomic DNA of bone marrow. Productof quantitative PCR was measured in an ABIPRISM 7900 HT sequence detection system(Applied Biosystems) similarly as previously
described.17, 18 Reactions were performed
with 100 ng of genomic DNA with SYBRGreen PCR Master Mix (Applied Biosystems)with the following primer set: HIV-U3
forward primer, 5’- CTG GAA GGG CTAATT CAC TC-3’, and HIV reverse primer,
5’- GGT TTC CCT TTC GCT TTC AG-3’.
The samples were normalized for mouse
gapdh with the following primer set: forward
primer 5’-CAT CAC TGC CAC CCA GAA
GAC-3’ and reverse primer 5’-TGA CCTTGC CCA CAG CCT TG-3’. A standardline for the lentiviral vector integrations wasdetermined from sorted mouse 3T3 cells

transduced at MOI of 0.05. Samples wereanalyzed with SDS2.2.2 software.
To quantify the number of lentiviral
integrations in human CD34+ cells, genomic
DNA was isolated using the QIAamp DNA
Mini Kit (Qiagen, Hilden, Germany) before
quantitative PCR detection using an ABIPRISM 7900 HT Fast (Applied Biosystems)
and qPCR core Kit (Eurogentec) using
the following primers: HIV-fwd1, 5’-TACTGA CGC TCT CGC ACC-3’, HIVrev1,
5’-TCT CGA CGC AGG ACT CG-3’.
The samples were normalized for humanactin with the following primer set: Actin
forward 5’-AGC GGG AAA TCG TGC GTG
AC-3’, Actin reverse 5’-CAA TGG TGATGA CCT GGC CGT-3’ (Metabion AG).
For quantitation of the integrated lentiviral
VCN, HeLa cells transduced at an MOI of
0.05 and subsequently FACS sorted were used
as control.

3

Statistical analysis
Statistical analysis was performed with
Graph Pad Prism (version 5.03). Significant
differences were determined by one-way
ANOVA on ranks with post hoc testing by
Dunn’s test or Mann-Whitney U test forcomparing two groups. Significant difference
was assumed if p<0.05.

| |Results
In vitro transduction efficiency byincreased cell and vector density
Transduction at higher cell and vector
concentrations with a constant MOI
improved transduction of mouse Linhematopoietic stem cell progenitors cells,
human UCB CD34+ cells and, to a lesser
extent, also rhesus macaque CD34+ cells(Fig. 1A). The total number of transduced cells
increased exponentially (Fig.1B). The vector
copy number (VCN) of the transduced cells ispresented in supplementary table S1.
Increasing the MOI in a fixed volume(increased vector concentration) improved
the transduction efficiency of rhesus macaqueCD34+ cells ~4-fold (Fig. 1C), and mouse55

Chapter 3

Figure 1. Cell and vector density improves HSC transduction efficiency. (A) Mouse Lin- cells,
rhesus BM CD34+ cells and human UCB CD34+ cells transduced at cell densities ranging from 2 ×
104 – 6 ´ 105/mL with an MOI of 1. The mean percentage GFP positive cells is increased at higher
cell densities (closed symbols SF.GFP and open symbols PGK.GFP lentiviral vectors, (N=1-3). (B)The mean absolute number of transduced cells increases strongly by higher cell densities and larger
number of cells (N=1-3). (C) The transduction efficiency of rhesus CD34+ cells at 6 ´ 105/mL and an
MOI 1 compared to MOI 37 (N=1). (D) An MOI of 1, but higher cell and vector densities increase
the transduction efficiency (N=1-2)

Lin- cells at low cell density (Supplementary
Fig. S1A). By keeping the MOI low (MOI 1),
but increasing the cell density, transduction
efficiencies of rhesus macaque CD34+ cells of~60% could be obtained (Fig. 1D).

56

In vivo reconstitution of high celldensity transduced hematopoietic
progenitors
We next investigated whether Lin- mouse
cells transduced under higher densities andtransduced at high and low MOI would
sufficiently reconstitute sublethally irradiatedCritical parameters for efficient HSPCs gene transfer

mice. High transduction efficiencies wereobtained in vitro (Supplementary Fig. S2A),
and long-term reconstitution was obtainedin primary and secondary recipients inerythrocytes, platelets and leukocytes(Supplementary Fig. S2B-G). Lin- cells werealso transduced at low MOI ranging from
MOI 0.25 to MOI 2. Similar ratios of GFP+
cells were observed in cultured transduced
cells and different blood lineages with a 3-foldincrease in GFP+ positive percentage between
MOI 0.25 and MOI 2 (Fig. S2H-K).

progenitors showed the same pattern ofincreasing transduction efficiency when celland vector density was increased (Fig. 2B).
A correlation between vector concentrationand transduction efficiency was also notedin the bone marrow and apheresis derived
CD34+ cells (Fig. 2C). Finally, the VCN of
the transduced hematopoietic progenitors
also correlated with cell density and varied
between 0.5 and 1.5 VCN per cell (Fig. 2D).

Transduction of humanhematopoietic progenitors fromperipheral blood, apheresis and bonemarrow samples with GLP grade
vector.

Strong activation of HSPCs improvestransduction efficiency but leads toprogenitor cell differentiation and loss oflong-term repopulating cells. To investigate
which culture parameters are important for
efficient transduction and reconstitution
under relatively high cell density, different
media (home-made SLM medium andStemMACS medium) and varied minimal
cytokine combinations were compared. Afterovernight transduction of Lin- cells and 7
days in vitro culture, similar transduction
efficiencies were observed when using SLM
or StemMACS medium, and a similar trend
could be observed in the MFI and VCNvalues (Fig. 3A-C). The combination of SCF,
TPO and Flt3-L (STF) resulted in the most
optimal transduction efficiencies, but single
cytokine additions resulted (SCF or TPO) in
high transduction efficiencies (Fig. 3A-C).
If transductions were performed with nocytokines, or Flt3-L alone this resulted in very
poor transduction (Fig. 3A-C). Colony assaysof Lin- cells seeded at day 1 were similar
between SLM and StemMACS, but 7 days
culture increased the number of CFU-GMs
specifically when cultured in StemMACS

To further investigate the effect of celldensities on transduction efficiency, humanCD34+ cells were subjected to different
transduction protocols including protocols
similar to those presently used in clinical
trials. A good laboratory practice (GLP) lot
was used for the experiments (Supplementarytable S2).
Pre-stimulated CD34+ hematopoietic
progenitors derived from buffy coats (Fig.
2A) were transduced at different cell densities.
A standard clinical protocol involving two
rounds of transduction at high MOI of 100
on RetroNectin-coated plates at 106 cells per
mL achieved transduction efficiencies of 89%.
A single round of transduction modified 79%
of CD34+ cells. Using a lower MOI of 10 atcell densities of 107 per mL resulted in GFP
positive percentages that approached those ofa single MOI 100 and 106 cells per mL (Fig. 2A).
Further experiments performed on apheresisand bone marrow derived hematopoietic

3

Cytokine combinations for efficienttransduction

57

Chapter 3

Figure 2. Human CD34+ cells transduced with GLP lentiviral vector lot. (A) CD34+ cells
were isolated from buffy coats and cultivated overnight at 4 × 105 cells per mL in StemSpan SFEM
medium supplemented with Flt-3L, SCF, IL-3 and IL-6. After 24-hour pre-activation, cell densities
were adjusted (105–107 cells per mL) before transduction at an MOI of 10 or 100. One sample wastransduced twice at an MOI of 100 on RetroNectin coated plates. The cells were analyzed 5 days
post transduction by flow cytometry. MFI (open bars) and percentage of GFP-modified cells (blackbars) are shown. C, control non transduced cells, N = 1 (B, C) Human CD34+ cells isolated from bonemarrow (BM) or leukapheresis samples (LP) were cultivated in CellGro SCGM medium supplemented
with SCF, TPO, IL-3 and Flt-3L at cell densities of 105, 106 or 107 cells per mL (N= 2-7) The cells weretransduced with an MOI of 10 or 20 (LP) or 10 (BM). (D) After 13 days of culture, transduced CD34+
cells from BM and LP were analysed for the VCN. Flow cytometric analysis and VCN was performed
5-6 days post-transduction (N=2-7).

medium (Fig. 3D and E). The cytokines did
not have significant effect on the number ofcolonies counted.
A similar experiment was performed
using human UCB CD34+ cells by comparing

58

STF and TPO conditions, in which
the transduction efficiency was comparable(Fig. 3F). StemMACS medium performed
slightly better with higher GFP percentage
and MFI, and progenitors derived from

Critical parameters for efficient HSPCs gene transfer

3

Figure 3. Effect of cytokine combinations on transduction efficiency. Different cytokine
combinations determine transduction efficiency of mouse and human HSPCs. SLM or StemMACS
HSC Expansion Media XF, human were tested in vitro presenting (A) GFP percentage, (B) mean
fluorescence intensity (MFI), and (C) vector copy number (VCN) per cell 7 days after transduction
at 106/ mL, MOI 2. N = 3 experiments. (D) Colony forming units- granulocyte monocyte (CFU-GM)
of Lin- cells seeded at day 1 and (E) 7 days of culture. N = 1-3 experiments. No = no cytokines, F= FLT3-L, T = TPO, S = SCF. (F) GFP percentage 7 days after expansion of human UCB CD34+ cells
transduced (2x106 per mL at an MOI of 10 in SLM or StemMACS media in combination with STF or

59

Chapter 3
TPO, (G) the MFI and VCN of SLM cultured cells and (H) CFU-GM and burst forming units- erythroid(BFU-E) of CD34+ cells seeded at day 1. N = 3 experiments. (I, J) Human CD34+ cells, isolated fromapheresis samples, were cultivated in CellGro SCGM medium or StemMACS medium supplemented
with SCF, TPO, IL-3 and Flt3-L at the indicated cell densities were transduced with an MOI of 100
at day 1 and analyzed on day 5 for GFP percentage or MFI (N=1). Data represent mean ± SEM.
Significant differences were determined by one-way ANOVA on ranks with by post hoc Dunn’s test,
p<0.05. SLM = standard lab medium.

Table 1. CD45.1 transduced Lin- cells for in vivo transplantation.

SLM + STF
SLM + SCF and TPO
SLM + SCF
SLM + TPO
SLM + No cytokines
StemMACS + STF
StemMACS + TPO

GFP+ %MFI

VCN/cellColonies/105 cells
Seeded day 1

57.1
52.9
41.8
24.7
6.4
67.9
33.4

10400
9239
7457
6956
5773
11284
8584

0.8
1.3
0.8
0.6
0.1
2.0
0.9

1375
1550
1675
1375
1025
1850
900

The GFP percentage, MFI and VCN/cell were determined at 7 days after transduction.

CD34+ cells seeded at day 1 resulted in similar
CFU-GM and BFU-E colony formation(Fig. 3F-H). Comparison with anothercommercial medium using human CD34+ cells
derived from apheresis material (Fig. 3I-J)
also supported the observation that
StemMACS medium resulted in improved
transduction efficiency of human cells.
To investigate whether mouse Lincells were able to efficiently reconstitutesublethally irradiated mice, transduced donor
Lin- cells were transplanted after overnighttransductioninmediumcontaining
selected cytokine combinations. Donor cells
reconstituted similarly to high levels in all
mice as measured in peripheral blood, BM

60

and spleen (Fig. 4A-C). SLM and StemMACS
medium with STF resulted in efficienttransgene expression in peripheral blood(Fig. 4D-F). This was confirmed in BM
and spleen (Fig. 4G-H). Interestingly, TPO
alone also resulted in high GFP percentages
if StemMACS medium was used, and nocytokines resulted in <5% GFP+ cells, but
did not have a significant negative effecton the reconstitution of transplanted cells(Fig. 4A, D-H). The use of the different
media did not affect the T, B and myeloid
lineage potential to reconstitute, and resulted
in similar transduction efficiencies in
the hematopoietic lineages, with SCF the least
efficient (Fig. I and J).

Critical parameters for efficient HSPCs gene transfer

3

Figure 4. In vivo reconstituted transduced Lin- cells transduced under different cytokine
combinations. Selected cytokine combinations were used to transduce donor mouse Lin- cells at
106/mL, MOI 2 in SLM or StemMACS (M) medium, and reconstitution of 1.4 × 105 cells transplanted
in recipient mice was monitored for up to 6 months (N = 2-6 mice/ group). The CD45.1 chimerismin (A) peripheral blood, (B) bone marrow (BM), and (C) spleen, and (D) the GFP percentage within
erythrocytes, (E) platelets and (F) leukocytes, and (G) BM and (H) spleen are shown. (I) contributionof T (CD3+), B (CD19+) and myeloid cells (CD11b+) distribution in CD45.1 donor peripheral bloodand (J) GFP percentage in the different leukocyte lineages.. Data represent mean ± SEM. Significant
differences were determined at 6 months after transplantation by one-way ANOVA on ranks by
Dunn’s post hoc test, p<0.05, n.s. not significant.

61

Chapter 3| |Discussion
HSPC clinical gene therapy trials use different
transduction protocols.9-12 These protocols areoften derived from older gRV transduction
methods, in which two or more transduction
rounds are included and 4-5 days of in vitro
culture is applied.3, 20 They also often includei) time to pre-stimulate CD34+ HSPCs witha cocktail of cytokines, ii) compounds such asprotamine sulfate and/or RetroNectin toenhance transduction and iii) the use ofhigh MOIs (MOI 25-200) .3, 9-11, 14, 20-22 Thismay have a negative effect on long-term
repopulating stem cells, may increase the riskof contamination, and can potentially alter
the integration profile, because certain
cytokine cocktails strongly promote cellcycling and dedifferentiation. Protocol
developments that result in minimizing the useof costly cytokines may be preferred. Adapting
protocols that require a reduced amount ofvector particles would be advantageous, since
every vector batch has to be validated for
quality and is generally laborious to produce.
Our aim was to test different HSPC (andvector) densities to enhance transduction
efficiency. Increased cell densities resulted insignificantly improved transduction of mouse
Lin- BM progenitor cells, rhesus macaqueCD34+ and human derived CD34+ HSPCs, sohigh MOIs are not required. Rhesus macaqueCD34+ progenitors are relatively difficult totransduce efficiently by HIV-derived lentiviral
vectors; hence transduction of these cells is
often performed with vectors based on simian
immunodeficiency virus (SIV).23-25 This iscaused by HIV-1 replication restriction at an
early post-entry step in simian cells, due tothe action of inhibitory cellular factors 26, suchas in part by the restriction factor tripartite
62

motif 5α protein (TRIM5α) 27 and bindingof the viral capsid to cyclophilin A. 25 Thisinitiated the development of chimeric HIV-1
vectors with SIV components to improvetransduction of rhesus cells. 28, 29 These
modifications increased transduction of
CD34+ rhesus macaque BM cells significantly,
thereby partially overcoming these barriers.
We have demonstrated that by increasing
the cell culture density of rhesus CD34+ cells
during vector exposure, up to 20% of cells
could be transduced using low MOIs, andup to 80% with high MOIs (from 0.25 to2), making in vivo experiments with HIV-1
lentiviral vectors in rhesus macaques, without
in vivo selection, feasible.
Cytokines are required for -RV
transduction of HSPCs. However, the useof cytokines for efficient LV transduction
may not be that critical. IL-3 has beenshown redundant for transduction of humanperipheral blood derived CD34+ HSPCs.30
Since IL-3 promotes differentiation ofhematopoietic progenitors,31 this can be easily
excluded from clinical gene therapy protocols.
Uchida et al 30 demonstrated that the use of
SCF alone reduced both the ability to engraft as
well as the transduction efficiency, in contrast
to earlier reports.32 On our hands, the useof SCF alone did not affect the repopulationof transplanted donor mouse cells (Fig. 4A
and I), but did not enhance the transduction
efficiency (Fig. 4 D-F, J). In our experimentswith SLM medium, adding TPO or SCF alone
improved transduction efficiency, similar
to an observation in human UCB CD34+
cells for SCF without other cytokines.32
Medium totally lacking in cytokines clearly
diminished transduction of short and long

Critical parameters for efficient HSPCs gene transfer

term repopulating cells establishing cytokines
are still required for transduction similarly toCD34+ UCBs 32 or mouse HSPCs.32 However,there are also some reports demonstratingthat cytokines can be omitted duringtransduction.33 The vital role of TPO in
the maintenance of HSPCs ex vivo and in vivo
has been previously demonstrated.34, 35 Our
experiments show that TPO alone, added
to StemMACS medium, aids transductionof both mouse and human HSPCs invitro, similar to SLM medium containing
the cytokines SCF, Flt3-L and TPO.
Amriche et al showed that low density
lipid receptor (LDL-R) was upregulated byaddition of ‘early acting cytokines’, which
was required for binding and reverse
transcription of vesicular stomatitis virus
G (VSV-G) pseudotyped LVs to resultin efficient transduction.35 Other factorsthat influence the susceptibility of HPSC
transduction that may be modulatedby the cytokines additions, for example
the cell cycle status at time of transduction8
or the antiviral proteasome activity able
to restrict lentiviral transduction,36 could
have played a role in the observed enhanced
transduction efficiency.
Haas et al have previously investigatedseveral parameters influencing transduction
efficiency of UCB CD34+ progenitors by
first and second generation LVs. Theirobservations were that vector concentration,
but not MOI, was directly related totransduction efficiency.37 Those experiments
were performed with relatively few cells(1-10 × 104 CD34+ UCB cells per condition)and thus by increasing vector concentration
at the same time that cell density wasincreased, transduction efficiency was
improved. However, transduction efficiencies

were lower than ours presumably due tothe relatively low cell densities used (max~ 105/mL). We demonstrated that by using
higher cell densities and larger numberof cells, clinically relevant transduction
efficiencies can be reached. Successfully upscaling these protocols could have significant
consequences in terms of amount of vector
required per patient, which in turn would
enable a future reduction of the LV genetherapy vector batch costs by up to a factor of
10. Few studies explored strategies to enhance
LV transduction of mouse and human
HSPCs, including the utility or rapamycin38
and recently, the application of microfluidictransduction system .39
Here, we show that cell and vector culture
density are critical components to enableefficient transduction of mouse, rhesus andhuman HSPCs, because these parameters
limits the conditions necessary to transduce
HSPCs, such as the amount of expensive
cytokines or the length of the transduction
protocol. Furthermore, we confirm theseresults with a clinical-grade good-laboratory
practice (GLP) vector batch on apheresis
derived human CD34+ HSPCs. Finally, wedemonstrate that by limiting the transduction
period to 20 hours with only the addition of
TPO, a high level of transduction is observed
and that, at the same time, the ability toreconstitute the hematopoietic system in miceis not affected.
In conclusion, we optimized the conditions
to efficiently transduce HSPCs. Our approach
can be used as a tool to perform preclinicalexperiments in rhesus macaques. The protocolpresented here is valuable for clinicalapplication as it would reduce the amountof vector significantly, limit the time of exvivo culture and increase the number of363Chapter 3

patients that can be treated by a single vector
batch, as well as reducing the amount ofexpensive growth factors required to culture
the hematopoietic stem cells.

| |Acknowledgments
The authors would like to thank Luigi
Naldini for the third generation LV vector
plasmids and Axel Schambach and Dr.
Christopher Baum for the mutated WPRE
element. This work was supported by
the European Commission’s 5th, 6th and 7th

Framework Programs, Contracts QLK3-CT2001-00427-INHERINET, LSHB-CT-2004005242-CONSERT and HEALTH-F5-2010261387-CELL-PID and by The Netherlands
Organization for Health Research ZonMw,
program grant 434-00-010.

| |Conflict-of-interest disclosure
CR and IJ are full time employees of Miltenyi
Biotec GmbH. CB is full time employee of
MolMed S.p.A..
Correspondence: Niek P. van Til,
Department of Hematology, Erasmus

64

University Medical Center, Dr. Molewaterplein
50, 3015 GE Rotterdam, the Netherlands;e-mail: N.P.vanTil@umcutrecht.nl

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Uchida N, Hsieh MM, Hayakawa J et al.
Optimal conditions for lentiviral transduction

Critical parameters for efficient HSPCs gene transfer| |Supplementary information
Supplementary table S1. Vector copy number cell density transductions.
Cell density (/mL)

Mouse Lin-

Rhesus CD34+

Human CB CD34+

2 × 104
6 × 104
1.2 × 105
2 × 105
6 × 105

0.13
0.33
0.92
1.13
0.99

0.02990.00140.46000.04500.4973

0.085
0.180
0.374
0.315
0.857

3

Average of N=2-3 experiments

Supplementary table S2. Composition of the GLP GFP vector batch.
Physical titer2,010 (ng p24 Gag/mL)

Functional titer
Infectivity
Infectious/total particles
Total residual DNA (Picogreen)
LTA residual DNA
Plasmid residual DNA
HCP
Sterility test
Endotoxin test

1.3 × 108 TU/mL
6.4 × 104 TU/ng p24 Gag
1:150
1.01 mg/mL
5.7 × 103 copies/mL
1.4 × 107 copies/mL
135 ng/mL
negative< 1.0 EU/mL(2.0 × 1010 pp/mL)A

pp are calculated assuming that 1 ng p24 Gag= 107 physical particle (pp)]
TU = transducing unit

A

Supplementary figure S1. Multiplicity of infection and cell density. An increase in MOI duringtransduction of mouse Lin- cells correlates with the percentage of GFP+ cells at a cell density of
5 × 104/mL (N = 1)67

Chapter 3

Supplementary figure S2. Long-term reconstitution of transduced cells in mice. (A)Percentage of GFP positive Lin- transduced cells at 1 × 106/mL after overnight and 6 days culture(N=1). Subsequently, different numbers of cells transduced at an MOI 2 or 30 were transplanted
in 6 Gy irradiated BALB/c mice. The in vivo reconstitution of transduced Lin- cells with GFP positive(B) erythrocytes, (C) thrombocytes and (D) leukocytes is presented including transplantationof ½ femur of primary into secondary recipients at month 7 (n=5-15, and secondary mice n =10-30 per group). Similarly, the mean fluorescence intensity (MFI) depicted for (E) erythrocytes,(F) thrombocytes and (G) leukocytes. Another experiment titrating down the MOI from 2 to 0.25
at 1.5 × 106/mL and transplanting different cell numbers, the in vitro results of (H) the percentage
of GFP positive cells and in vivo reconstitution with secondary transplantations presenting (I)erythrocytes, (J) thrombocytes and (K) leukocytes.

68Preclinical efficacy and safety evaluation ofhematopoietic stem cell gene therapy ina mouse model of MNGIE
Rana Yadak1,2, Raquel Cabrera-Pérez3, Javier Torres-Torronteras3,
Marianna Bugiani4, Joost C. Haeck5, Marshall W. Huston1, Elly Bogaerts6,
Steffi Goffart7, Edwin H. Jacobs 6, Merel Stok2,6,8, Lorena Leonardelli9,
Luca Biasco9,10,11, Robert M. Verdijk12, Monique R. Bernsen5,
George Ruijter6, Ramon Martí3, Gerard Wagemaker*2,13,14,
Niek P. van Til*2,15, Irenaeus F. M. de Coo*1
Department of Neurology, Erasmus University Medical Center, Rotterdam, The Netherlands; 2Department
of Hematology, Erasmus University Medical Center, Rotterdam, The Netherlands; 3Research Group on
Neuromuscular and Mitochondrial Diseases, Vall d’Hebron Institut de Recerca (VHIR), Universitat Autònoma
de Barcelona, and Biomedical Network Research Centre on Rare Diseases (CIBERER), Barcelona, Catalonia,
Spain; 4Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands; 5Departmentof Radiology & Nuclear Medicine, Erasmus University Medical Center; 6Department of Clinical Genetics,
Erasmus University Medical Center; 7 Department of Biology University of Eastern Finland, Joensuu, Finland;
8
Department of Pediatrics, Erasmus University Medical Center, Rotterdam, The Netherlands; 9San Raffaele
Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy; 10Gene Therapy Program, Dana-Farber/BostonChildren’s Cancer and Blood Disorders Center, Boston, MA, US; 11University College of London (UCL), Great
Ormond Street Institute of Child Health (ICH), London, UK; 12Department of Pathology, Erasmus University
Medical Center; 13Hacettepe University, Stem Cell Research and Development Center, Ankara, Turkey; 14Raisa
Gorbacheva Memorial Research Institute for Pediatric Oncology and Hematology, Saint Petersburg, Russia;
15
Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht, The Netherlands.
1(Submitted)

4Chapter 4| |Abstract
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal
recessive disorder caused by thymidine phosphorylase (TP) deficiency resulting in systemic
accumulation of thymidine (d-Thd) and deoxyuridine (d-Urd) and characterized by early
onset neurological and gastrointestinal symptoms. Long-term effective and safe treatment is
not available. Allogeneic bone marrow transplantation may improve clinical manifestations,but carries disease and transplant related risks. In this study, lentiviral vector basedhematopoietic stem cell gene therapy (HSCGT) was performed in Tymp-/-Upp1-/- mice withthe human phosphoglycerate kinase (PGK) promoter driving TYMP. Supranormal blood TP
activity reduced intestinal nucleoside levels significantly at low vector copy number (median,1.3; range,0.2-3.6 ). Furthermore, we covered two major issues not addressed before. First, wedemonstrate aberrant morphology of brain astrocytes in areas of spongy degeneration, which
was reversed by HSCGT. Second, long-term follow-up and vector integration site analysis
were performed to assess safety of the therapeutic LV vectors in depth. This report confirms
and supplements previous work on the efficacy of HSCGT in reducing the toxic metabolites
in Tymp-/-Upp1-/- mice, using a clinically applicable gene transfer vector and a highly efficientgene transfer method, and importantly demonstrates phenotypic correction with a favorable
risk profile, warranting further development towards clinical implementation.

72

HSCGT in a mouse model of MNGIE| |Introduction
Mitochondrial
neurogastrointestinal
encephalomyopathy(MNGIE)isanautosomal recessive disease caused bymutations in the thymidine phosphorylase
gene (TYMP)1, which result in partial orcomplete deficiency of the enzyme thymidine
phosphorylase (TP)2. TP deficiency resultsin systemic accumulation of the substrates
d-Thd and d-Urd in plasma and tissues of
MNGIE patients3 and thereby to imbalances
in intra-mitochondrial deoxyribonucleoside
triphosphates pools (dNTPs), which has been
considered to induce somatic mitochondrial
DNA alterations in tissues of MNGIE
patients4, 5. The clinical spectrum of MNGIE
ranges from mild symptoms in late-onset
cases6 to early teenage onset with fatal
malabsorption and intestinal dysmotility7.
The typical symptoms include ptosis andophthalmoplegia, gastrointestinal dysmotility,
neuropathy with reduced sensory-motor
conduction, as well as central nervous systemanomalies4, 8. The latter manifest on brain MRI
as a unique pattern of progressive symmetricalcerebral (and occasionally cerebellar) hyperintense signal changes on T2-weighted
images9. The available treatment options for
MNGIE such as peritoneal dialysis, platelets
infusion and enzyme replacement therapy failto achieve persistent clinical improvement10-12.

Asalong-term

hematopoietic

stemsolution,cellallogeneictransplantation(HSCT) has been proposed; however, this isassociated with high mortality rates due todisease and transplant related complications13,
14. Recently, liver transplantation has emerged

as a promising treatment option preferably
for patients with pre-existing liver failure;
this however requires a matched organ
donor and long-term immunosuppression15.

4

Autologous hematopoietic stem cell genetherapy (HSCGT) could be beneficial for
MNGIE patients as a single intervention
with low-dose conditioning and poses norisks of graft rejection or graft- versus hostdisease. Third generation self-inactivating
lentiviral (LV) vectors are currently usedsuccessfully in clinical trials for othermetabolic disorders16-18. The present study
supplements and confirms previous reports 19,
20

by using (i) a highly efficient transduction

method, (ii) clinically applicable LV vectors
to evaluate the therapeutic efficacy to reverse
the biochemical phenotype, and extends thesereports by demonstrating (iii) therapeutic
efficacy of HSGCT in reversing pathologicalchanges, particularly those of the brain, andassessing (iv) potential adverse effects such as
pheno- and genotoxicity.

| |Results
LV vectors, experimental design, TP
expression and enzyme activity in target cells.
Similar to previous studies19-21, Tymp-/-Upp1-/mice (referred to here as KO) were used.
Unlike in humans, murine UPP1 degrades Thd
and d-Urd, therefore Tymp-/-Upp1-/- , rather
than Tymp-/- mice22, reflect the biochemical

phenotype of MNGIE patients. A LV vector
containing both TP and GFP sequences(PGK-TP-GFP) was reported previously19
for biochemical correction in Tymp-/-Upp1-/mice and is used in this study as a control.
In the present study, third generation
self-inactivating LVs containing a similar
73

Chapter 4

backbone as currently used in clinical trials
for metachromatic leukodystrophy (MLD) 17
and Wiskott-Aldrich syndrome23 wereconstructed for expression of TP. The humancellular phosphoglycerate kinase (hPGK)
promoter was used to drive expression of
the native human TYMP sequence (TP) as
well as the re-coded human TYMP sequence(TPco) in the therapeutic vectors, as wellas to drive expression of the GFP sequencein the sham control vector (Figure 1A).
The rationale for using TPco was to enhanceprotein production with a minimum numberof transduced donor cells and vector
copy number, as reported for IL2RG and
RAG2 24, 25. The strong spleen focus forming(SF) virus promoter, which may contributeto leukemic events by transactivating protooncogenes26, was used to evaluate potential
phenotoxicity by excessive overexpressionof TYMP and/or genotoxicity rather thanan option for potential clinical application(Figure 1A).
Two virus concentrations (multiplicity of
infection, MOI) were used for transduction,i.e., MOIs of 3 or10. By transplantation
experiments (Figure 1B) the efficiency of
the therapeutic LVs (PGK-TP, TPco) were
evaluated for (i) biochemical correction (MOI
3 or 10) and (ii) neurological correction asassessed by brain magnetic resonance imaging(MRI), pathology and behavioral tests (MOI10). To assess the safety of HSCGT primary
recipients were subjected to long-term follow
up for potential transgene overexpressionrelated phenotoxicity (in particular in
the SF-TPco treatment group) and LV- related
genotoxicity, as well as an integration siteanalysis of gene modified cells prior to andafter engraftment in primary recipient mice

Figure 1. Lentiviral vectors and design of transplantation experiments and TP expression
in the target cells. (A) Third-generation self-inactivating LV vectors used in the study and theirelements are shown. pCCL and pRRL, LV vectors containing CMV-HIV or RSV-HIV 5’ long terminal
repeats respectively; RRE, Rev response element; cPPT, central polypurine tract; hPGK, humanphosphoglycerate kinase promoter; hTP, native coding sequence of the human TYMP cDNA; hTPco,codon-optimized sequence; IRES, internal ribosome entry site; GFP, green fluorescent protein; SF,spleen focus forming virus promoter; WPRE, Woodchuck hepatitis posttranscriptional regulatory
element; bPRE4*, adapted form of WPRE38. (B) Design of HSC transplantation experiments.
The hematopoietic stem and progenitor cells (Lin-) from male donor mice were ex vivo transduced
over night by the LV vector constructs and transplanted into female recipient mice. To determine
the integration site profiles, LAM-PCR and integration site analysis were performed on a fraction of
the transduced cells in vitro and BM cells from transplanted primary mice in vivo; see also Figure S3A.
To evaluate LV vector-related insertional transformation in vivo, Lin- cells from each primary recipientwere transplanted into two secondary KO female recipient mice. All transplanted mice received 6Gy
total body irradiated 24hr prior to transplantation. All experiments included age matched untreated
KO and WT controls. a End point analysis: sample collection for biochemical and molecular analysis,
flow cytometry analysis. MRI= magnetic resonance imaging, IHC = immunohistochemistry. (C-E) TP
expression and activity in KO Lin- cells transduced (MOI 10) and cultured in vitro. (C) qPCR analysis
of WPRE mRNA expression normalized to GAPDH levels and corrected for VCN/ cell. The horizontal
line represents the median, n= 3-4 replicates. (D) Western blot of total protein extracts from
Lin- cells and their content corrected for VCN/ cell, n=1, n.a.= not applicable. Included are alsolysates of SF-TPco treated cells confirming the higher TP protein with this promoter. (E) TP enzyme
activity, n=1.

74

HSCGT in a mouse model of MNGIE

4

aged 13 months, i.e., 11 months after HSCGT,
to estimate the risk of insertional oncogenesis,
and finally by secondary transplantation,a useful pre-clinical tool to detect vector
induced hematological transformations
Codon

optimizationof26, 27

TYMP

.

did

not result in improved WPRE mRNA, TP
protein levels and TP enzyme activity (Figure
1C-E); therefore the two vectors were usedinterchangeably and referred to as PGKTP(co) hereafter.

Biochemical correction andmolecular chimerism following exvivo HSCGT
Stable blood TP activity was observed bothin the PGK-TP-GFP group as previously
reported20, and in recipients of the therapeutic
75Chapter 4

LV- PGK-TP(co) (Figure 2A). Lin- cells
successfully engrafted both PGK-TP(co) and
PGK-GFP recipient KO mice and integrated
vector copies were detected (Figure 2B).
We next measured TP activity in diseased
tissues of our mice. Transduction resulted
in increased TP activity to normal levels inbrain and small intestine in the PGK- TP(co)treated mice (Figure 2C). As a consequenceof the high TP activity, urine and plasma
nucleosides were undetectable in almost allrecipients of PGK-TP(co) with extensive
reduction in brain nucleosides compared toboth KO and WT animals (Figure 2D and
Figure S1A). Overall, median nucleosidelevels were reduced insufficiently at the lower
MOI (Figure 2D and Figure S1A), (MOI
3, range, 0.1-0.8 VCN/ cell and percentage
chimerism 47 -76 %, n= 5, data not shown).
Eleven months after transplantation,
recipients of PGK-TP(co) displayed 98% and
57% reduction in median intestinal d-Thd
and d-Urd levels, respectively (Figure 2E andFigure S1B). Moreover, HSCGT provided
high TP enzyme activity and reduced
nucleoside levels in skeletal muscle and liver(Figure S1C). Altogether, although at an MOI
3 significant reduction of nucleosides wasobserved, full correction of the biochemical
phenotype, only occurred at transplantationof 6 Gy irradiated recipients of 5×105 MOI
10 transduced cells, particularly in the small
intestine , in contrast to the previous study20.

MRI and rescue of brain white matterdamage after HSCGTNeurological changes are consistently
reported in MNGIE patients, and minorchanges were reported in old Tymp-/-Upp1-/mice aged ≥18 months22. To characterize
neurological abnormalities at earlier ages
76

and to address the impact of HSCGT as
early as at 2 months of age, brain MRI
scans and immunohistochemistry (IHC)
were performed. In contrast to WT mice,an increase in percentage of fluid content
was observed as a hyper-intense signal in
the brains of 6 to 12 month aged KO (5 outof 8) (Figure 3A). Increased T2 signals areindicative for fluid accumulation in the whitematter surrounding the lateral ventricles,
suggesting edema. White matter edema
was absent in most treated mice; 8 out of 10
treated mice fell in the same hyper-intensevolume range of WT animals (Figure 3B).
Brain sections from KO mice stained withmature myelin proteolipid protein (PLP)and myelin basic protein (MBP) revealed
the presence of progressive vacuolizationin the cerebellar and cerebral white matterstructures absent in WT mice. White mattervacuolizations were lined and crossed by thinimmunopositive tissue stands, consistent withintramyelinic edema (Figure 3C, D). PLP and
MBP-staining revealed disappearance of whitematter vacuolization in the cerebellar whitematter and corpus callosum of PGK-TP(co)treated group (Figure 3C, D). IHC staining
for the astrocyte-specific glial fibrillary acidic
protein (GFAP) revealed increased thicknessof astrocyte processes abutting the bloodvessels in KO mice as young as 2 months,
compared with age matched WT mice, which
was further increased in 12 months aged KO
mice (Figure 3E, F). The thickness of astrocyte
perivascular processes in cerebellum, corpus
callosum and hippocampus white matterof PGK-TP(co) treated mice were reduced
to normal levels (P< 0.0001) (Figure 3E, F).
In addition, GFAP staining of the cerebellar
cortex revealed that Bergmann glia werelocalized to the molecular layer in KO mice

HSCGT in a mouse model of MNGIE

4

Figure 2. Long-term biochemical correction and molecular chimerism following HSCGT. Lincells were transduced with the therapeutic and control LV vectors at MOI 10 and 5×105 cells weretransplanted into 6 Gy pre-conditioned 2-month-old KO mice. (A) TP enzyme activity was measured
in blood at months 1 and 11 after transplantation, n=3-9mice/group. (B) BM cell chimerism andvector copy number of recipient mice, n=5-9 mice/group. (C) Brain and intestine TP activity were
measured 11 months after transplantation, n=3-12 mice/group. (D) Quantification of Thd in urine,
d-Thd in plasma, brain 8-11 months after transplantation (MOI 10, 3), n=4-11 mice /group and (E)in intestines 11 months after transplantation, n=3-9 mice /group. The horizontal line represents
the median, *P <0.05, **P <0.01 and ***P <0.001, n.s. = not significant.

77

Chapter 4

in contrast to the normal localization in
the Purkinje cell layer in WT mice and after
treatment (Figure 3G). Altogether, brain MRI
and IHC revealed the classical morphologicalchanges in KO mice, however at a younger
age and further demonstrated an underlying
edema linked to changes of the brain
microglia, which are reversed upon HSCGT.

Safety evaluation
Excessivetransgene
expression-related
phenotoxicity and/or genotoxicity caused
by LV insertional mutagenesis are potential
drawbacks for clinical application of HSCGT.
These two issues were not addressed inprevious studies 19,20 of HSCGT for MNGIE.

A total of 106 controls and primary recipientsof 5×105 Lin- cells transduced with therapeuticand GFP LV were monitored for 11 months.
Some recipients of PGK-TP or TPco weresacrificed and analyzed prematurely, becauseof death or high discomfort scores. We
have been unable to determine the actual
cause of wasting, but importantly, vector
positive hematological clonal expansion
was not observed (Table 1). At termination,
hematopoietic reconstitution between groups
was similar, except for a reduction in numbersof spleen CD3+ T-cells in PGK-TP(co) andperipheral blood CD19+ B-cells in SF-TPcotreated mice (Figure 4 and Table S1).
Nucleosides were not detectable in urine andFigure 3. Brain magnetic resonance imaging, characterization of brain white matterdamage and rescue by HSCGT. Lin- cells were transduced with the therapeutic LV vectors at
MOI 10 and 5´105 Lin- cells were transplanted into 6 Gy pre-conditioned 2-month-old KO mice.
MRI scans were performed on 2- month-old (young controls) and 6-12-month-old controls andtreated mice. (A) Representative T2 scans of WT, KO and treated mice. Hyper-intense voxels
within the brain (white dotted) were quantified by using a reference ROI (red dotted). (B) Resultsof the quantification of cerebrospinal fluid (CSF) and white matter edema. The data displayedis the percentage of voxels with high signal intensity of the whole brain, n= 6-10 mice/group.
The horizontal line represents the median. Immunohistochemical staining was performed on brain
tissue collected from (young controls) and 12-month-old controls, n= 2-3 /group and treated mice
9-10 months after transplantation, n= 4 mice. (C) Staining for the mature myelin protein proteolipid
protein (PLP) of cerebellum of young (left panel and right panels at higher magnification) and old(middle panel) KO and WT female mice shows normal myelin amounts in the cerebellar white matterof a PGK-TP treated mouse. Quantification shows significantly more vacuoles in the cerebellar
white matter of KO animals. The horizontal line represents the median, *P<0.05, **P <0.01 and***P <0.001. (D) Staining for the mature myelin protein myelin basic protein (MBP) of the corpus
callosum shows myelin pallor in KO compared to WT mice, which is rescued after gene therapy intreated female mice; a section from a recipient of PGK-TPco transduced HSCs is shown. (E) Staining
for the astrocyte-specific intermediate filament protein glial fibrillary acidic protein (GFAP) shows
thicker cellular processes abutting the blood vessels in the cerebellar white matter of KO mice as
young as 2 months compared to WT mice. The asterisk indicates the blood vessel lumen. Labelingagainst GFAP shows thinner perivascular astrocytic processes in the brain of a recipient of PGK-TPcotransduced HSCs. (F) Thickness of astrocyte processes surrounding blood vessels, n≥56 processes/
group. The horizontal line represents the median, ***P<0.0001. (G) Stain against the astrocytespecific intermediate filament protein glial fibrillary acidic protein (GFAP) shows that Bergmann glia
(arrows) are located in Purkinje cell layer (PCL) of the cerebellar cortex of WT and a recipient mouseof PGK-TPco transduced HSCs (left and right panels respectively), whereas in KO mice they are
mislocalized to the molecular layer (ML) (middle panel), GL; granular layer.

78

HSCGT in a mouse model of MNGIE

plasma and extremely reduced in the brainsof SF-TPco mice as expected due to excessive
increase in blood TP activity (Table S2).
Although percentage donor chimerism in

SF-TPco treated mice was high (Table S2),
twelve out of 14 primary recipients weresacrificed prematurely mainly due to severe
body weight loss (>15%) observed at death479Chapter 4

Figure 4. Hematopoietic reconstitution after HSCGT. The following analyses were performed
9-11 months after transplantation of Lin- cells transduced at MOI 10. (A) FACS analysis of peripheral
blood, bone marrow and spleen cells. Cells were stained with antibodies against CD45.2 and CD3,
CD19, CD11b for detection of respectively, T, B and myeloid leukocytes (n= 3-12 mice/group),
the horizontal line represents the median, *P <0.05, **P<0.01. (B) Colony-forming unit assay. BM
cells were cultured and granulocyte-monocyte progenitors (CFU-GM) were counted, n= 1-6 mice /group. The horizontal line represents the median.

80

HSCGT in a mouse model of MNGIE
Table 1. Hematological aberrations and clonal expansion of transduced cells were not observed inprimary recipient mice.

LV vector

Number of vectorpositive
hematologicalaberrations a

Number ofnon-vector-positive
hematologicalaberrations b

Number of prematurely
dead mice or micesacrificed with highdiscomfort scores c

WT CTR (n= 13)
KO CTR (n=18)
PGK-GFP d

0
0
0

0
0
0

0
1 (macrocephaly)
1 (found dead)(n= 25)
PGK-TP-GFP (n=6)
PGK-TP (n=19)

1 (rectal prolapse)
0
0

0
0

1 (passive)0
1 (plagiocephaly)

4

1 ( wounds)
1 (passive)
1 (epileptic seizure
during behavioral test)
2 (found dead)
PGK-TPco (n=11)00

1 ( body weight loss) e
1 (epileptic seizure
during behavioral test)
1 (found dead)SF-TPco (n=14)002 (passive)
5 (body weight loss )1 ( kyphosis, body weightloss)
2 (passive)
1 (edema)
1 (found dead)2 (kyphosis)

Mice were transplanted with LV transduced 5×105 BM Lin- cells (MOI 10) and followed for 11 months
after transplantation. CTR, control untreated mice. a,b Hematological aberrations and clonal expansion oftransduced cells were assessed based on altered FACS phenotypes and/or elevated WBCs, and /or enlarged
spleen, thymus or lymph nodes. BM was tested for male donor cells and vector copy number. c The rightcolumn includes mice that were found dead or mice sacrificed prematurely during the experiment due
to high discomfort scores and hematological aberrations could not be identified (details are specified
between parenthesis). d PGK-GFP group include recipient mice of KO or WT Lin- cells transduced by
PGK-GFP. e Body weight loss at the time of death >15% over a time span of 1 to 2 months.

(n= 6 out of 12). This increased incidence of
weight loss was not observed in PGK-TP or
TPco treatment groups (Table 1 and Figure S2).
To evaluate genotoxic risk, we analyzed
the therapeutic and GFP containing LV

vector integration site profiles in mouse Lincells in vitro and in BM cells from primary
recipient mice (Figure 1B and Figure S3). In
total, 7435 unique integration sites (IS) wereretrieved by LAM-PCR and sequencing from81

Chapter 4

4 in vitro samples and 10 transplanted mice
(Table 2). A polyclonal integration pattern
was observed with 1174 common integrationsites (CIS)28. In total, 225 IS near oncogenes
were identified, accounting for 3.03% of all
genes (Table 2). We analyzed the IS patterns
for the oncogene frequency between groups
classified based on promoter, i.e., PGK vs.
SF, or pre-transplantation lineage negative
cells vs. bone marrow in vivo or transgene
GFP vs. TP (co), but no differences wereobserved (Table 3). To assess genotoxicity
further, BM Lin- cells from primary recipients(n= 44) were transplanted into 88 secondary

recipients. In both the primary and secondary
recipients, we did not observe any vector
positive hematological clonal expansion
in the therapeutic PGK-TP or PGK-TPcogroups (Table S3). In nine recipients of cells
LV transduced by PGK-GFP, TP, TPco and
SF-TPco vectors altered leukocyte levels andcounts revealed hematological aberrations.
These cases had very low median VCN/
cell (0.03 with range 0.01-0.07) and donor
chimerism (0.7% with range 0.2-7.1%, n= 8,
Table S3) and therefore were classified as nonvector-related. Two PGK-TP-GFP recipientsof Lin- cells of a single primary mouse

Table 2. Summary of LV vector integration sites (IS) retrieved by LAM PCR and sequencing
Cell type(donor-recipient)
In vitro
Lin- (KO)Lin- (KO)In vivo
BM (WT-WT)BM (KO-KO)
BM (KO-KO)

LV vector

Number of Number Number of %samplesof IS
oncogenes Oncogenes

PGK-TP
SF-TPco

2
2

3147
2489

102
74

3.24
2.97

PGK-GFPPGK-TP
PGK-TP-IRES-GFP
TOTAL

2
5
3
14

782
488
529
7435

20
3
26
225

2.56
0.61
4.91
3.03

Table 3. Summary of LV integration sites (IS) classified based on promoter, cell type and transgene

Group

CommonNumberNumber Unique integration Unique
Number of %of Samples of IS
genes sites (CIS) oncogenes Oncogenes Oncogenes

All
14
PGK only
12SF
2Lin- (in vitro) 4
BM (in vivo) 10
GFP only2
TP (co)
12

82

7435
4946
2489
5636
1799
782
6653

4104
3082
1869
3408
1379
666
3799

1174
702
241
815
178
48
1030

110
87
52
102
35
17
106

226
152
74
177
49
20
206

3.03
3.07
2.97
3.14
2.72
2.56
3.10

HSCGT in a mouse model of MNGIE

developed vector-positive diffuse intermediate
to large B220+GFP+ B-cell lymphoma (Table
S3) at 9 and 11 months after transplantation.
The VCN/ cell and percentage chimerism
were 0.86 and 67% in one mouse and 0.76
and 48% in the other mouse. An abundanceof abnormally large B cell population of
B220+GFP+ cells was observed in BM (Figure
S4A) of both mice, i.e., > 80% of total BM
cells, (Table S3), spleen and peripheral bloodof one mouse. Pathological analysis showed
infiltration of malignant B cells in spleen
and liver (Figure S4B). The B-cell clone
contained a single dominant integration site(IS) in the chromosome 11 gene Zfp207. No
known oncogenes were found within 500kb
of this IS (Figure S4C). Of note; the primary

donor mouse had two dominant IS located in
the genes Aplp2 on chromosome 9 and Csmd3
on chromosome 15, but the Zfp207 IS was notdetected by LAM-PCR. Primer specific PCRamplification confirmed very low presence of
this IS in bone marrow and spleen (data notshown). Besides the miscellaneous symptoms
observed in therapeutic PGK-TP(co) treatedmice, which are not likely to be related to
the gene therapy procedure, phenotoxicity
was observed in the SF treatment group, i.e.

4severe weight loss in the majority of the treatedcohort.

Hematologicalabnormalities

orincreased risk of insertional mutagenesis were
not observed in the group of mice receiving
the therapeutic vectors.

| |Discussion
The present study, using a clinically applicable
SIN LV vector with a modified WPRE andwithout GFP and a highly efficient, overnight
gene transfer procedure, demonstrates
not only full biochemical correction, but,importantly, also complete clearance ofintestinal nucleosides and a remarkablephenotype correction in the brain of Tymp/Upp1-/- mice. Overall, the biochemicalcorrection obtained in blood and tissues
reported earlier by using PGK-TP-GFP 20
was confirmed here in a larger cohort of miceby using the therapeutic vectors LV-PGKTP(co). The additional complete clearance
of intestinal nucleosides and remarkablephenotypic correction in the brain
demonstrates the feasibility of a curative genetherapy approach for MNGIE patients.
In MNGIE patients, the intestines areseverely affected, resulting in cachexia, severe
infections and death. The intestine facilitates

uptake of nutrients possibly requiring highTP enzyme activities to reduce incoming
thymidine levels locally. Of note, intestinal TP
activity in WT mice is substantially higher thanthat in blood and brain tissue (Figure 2A, C),
which emphasizes that high TP activity levels
in intestine are likely required to normalize
nucleosides. In our study, therapeutically
relevant transduction efficiencies by PGKTP(co) ( median VCN/ cell= 1.3 ( range,
0.2-3.6, Figure 2B) resulted in complete
clearance of intestinal nucleosides, in contrast
to the earlier study20 in which d-Thd wassignificantly reduced in plasma and brain, but
not in the intestine. In addition to the highliver and muscle TP activity, this finding of
an improved biochemical correction supports
our low vector MOI (3, 10 vs.10020) and pretransplant conditioning protocol.
The current experiments, performed
with high transduction efficiency and dose,83Chapter 4

resulted in supranormal levels of TP activity
and in near complete elimination of systemic
nucleosides, i.e., below WT levels, indicatingthat lower enzyme levels should be sufficient
to correct the biochemical imbalance. For
gene modified and vector dose titration
experiments relatively large cohorts of miceare required to demonstrate a dose response
relationship. However, whether or not lower
cell and LV dose would be efficient is of doubt,
since the small intestine requires a high celldose and an MOI of 10 to normalize TP
activity and nucleosides (Figure 2C, E, S1B).
The absence of a clear clinical phenotype
is a limitation of the only murine diseasemodel of MNGIE, and very few pathologicalfindings have been reported, mainly in
Tymp-/-Upp1-/- mice aged ≥18 months22. In
the present study, we observed pathologicalwhite matter changes in KO animals as early
as at 2 months of age and MRI changes at 6
months of age, which resembled findingsin the brain of MNGIE patients, i.e., edema
without demyelination or gliosis andhyperintense T2-weighted signal changes8,
29. Importantly, HSCGT resulted in elevatedTP activity restoring brain white matter
integrity and reducing edema as evident by
MRI and IHC. Another study of MNGIE
patients demonstrated that loss of brain TP
was associated with loss of blood brain barrier
integrity, which resulted in activation ofastrocytes30. We showed increased astrocytic
process thickness in Tymp-/-Upp1-/-mice,
which is limited to the cell processes abutting
blood vessels, which argues against reactive
astrogliosis and rather suggests swellingof the astrocyte perivascular end-feet.
Such a phenotype might be responsible
for the observed progressive intramyelinic
edema in Tymp-/-Upp1-/- mice as reported in84

other white matter diseases31 and could be
explained by an interference with the normal
function of astrocytes in maintaining
ion-water homeostasis32. The latter finding
argues for further research to investigate
the molecular mechanisms underlining brain
pathophysiology in MNGIE.
We did not demonstrate TP positive
hematopoietic cells in the brain, however,previous murine gene therapy studies using
AAV2/8 vectors21 transducing the liver
showed reversal of nucleoside imbalance
without the local presence of TP in the brain.
However, in HSCGT, gene modified monocyte
progenitors may migrate to the brain anddifferentiate into microglia.33 Since TP isa cytoplasmic protein, we measure this TP
activity in the brain (Figure 2C), indicating thattransduced microglia might reside in the brain
after long-term follow-up. Despite the brain
pathology in Tymp-/-Upp1-/- mice , we could
not detect significant differences in several
memory and motor function assessments,
which involved cerebellum, cortex andhippocampus, between the Tymp+/+Upp1+/+,Tymp-/-Upp1-/- and gene therapy treated mice(data not shown). This could be attributed todifferences in physiology and life span between
mice and man. Other leukodystrophy mousemodels, including those for megalencephalicleukoencephalopathy
withsubcorticalcysts similarly lack a clinical phenotype
despite the presence of severe pathologicalabnormalities31. Similar to the analysis of
MNGIE human brains30, we did not detect
mtDNA depletion, deletions or any changes in
mtDNA topology or strand breaks. Although
the variation between individual mice is large,
Tymp-/-Upp1-/-and treated mice showed signsof replication stalling in brain tissue (data notshown), which requires further investigation.

HSCGT in a mouse model of MNGIE

Of major concern, as yet unaddressedis the potential transgene or LV- related
phenotoxicity
orgenotoxicity.
Moreexperiments are required to elucidate
the nature of the toxicity observed in the SF
treatment group. We did not observe loss of
vector-transduced cells in the hematopoietic
compartment, so overexpression has no direct
effect on hematopoietic cells. Additionally,
the toxicity is not related to the LV vector
since LV-PGK-GFP did not show toxicity(Table 1 and Figure S2). Therefore, we
assume that the observed toxicity is related
to the excessively expressed TP by the strong
SF promoter, the increased mortality likely
due to complete elimination of d-Thd and/or
d-Urd affecting downstream mitochondrial
dNTPs. dNTPs pool imbalance triggers
cell cycle arrest and apoptosis in vitro inhematopoietic cell lines and yeast variants34, 35,
which, however, needs further investigationin Tymp-/-Upp1-/- mice. Using the therapeutic
vectors, oncogenic adverse effects were notobserved after a total follow-up in primaryand secondary recipient mice of 22 months,
demonstrating the safety profile of PGKdriven TP expression. A single, possibly
insertional, oncogenic event was observedin the PGK-TYMP-IRES-GFP treated mice.
Two secondary recipients acquired the same
B-cell lymphoma clone with the PGK-TPGFP vector integrated near the Zfp207 gene,which could be traced back to one primary
recipient mouse. Zfp207 or its humanhomolog is not reported as a proto-oncogene,but has a role in cell cycle and mitotic nuclear
division: in particular, ZFP207 is requiredfor proper chromosome alignment36 andZfp207 knockdown has been associated withlethal chromosome congression defects intransformed cells37. The B-cell lymphoma

contained a lentiviral integration with an
early version Woodchuck hepatitis virus
PRE element. Although reports have nevershown unequivocally that the use of sucha PRE element carries additional insertional
oncogenic risks, our therapeutic lentiviral
vectors contain a mutated element (bPRE4*)
to reduce risks further38. Since the lymphomas
in the secondary transplanted mice wereidentical, the oncogenic event necessarily
occurred in the primary transplanted mouse.
The integration near the Zfp207 gene might
have contributed to the development or
expansion of this clone, but it is not excluded
that the observed lymphoma is not related
to vector integration at all, which would
require further investigation of potentially
oncogenic mutations.
In summary, the therapeutic PGK-TP(co)vectors provided sufficient expression forbiochemical and phenotypic correction
without detectable phenotoxicity anda favorable safety profile, similar to the LV
vectors currently in clinical trial for otherinherited disorders. Nonetheless, excessive
TP expression in the LV-SF treatment group,
not intended for clinical application, resulted
in reduced survival (Table 1), a finding inneed of further investigation and underlining
the requirement of a moderately strong
house-keeping gene promoter such as PGK
for clinical application in MNGIE, possibly
in combination with low-intensity or nonmyeloablative conditioning. It is concluded
that the present study warrants further
development including cell and vector dose
response experiments with a larger cohort ofmice, biodistribution studies and assessmentin human hematopoietic cells, aiming
at clinical implementation of a curativeintervention therapy for MNGIE.

485

Chapter 4| |Materials and Methods
Lentiviral vectors
The self-inactivating transfer vector (SIN)
pCCL- hPGK-TP-IRES-GFP-WPRE wasdescribed before, and is used in our study as
a control19. The human TYMP sequence fromthis LV vector, a re-coded TYMP sequence(human TPco, GenScript, Piscataway, NJ,
USA)24 with optimized open reading frame(Genscript algorithm) with a consensus Kozak
sequence and an additional stop codon, andthe green fluorescent protein GFP sequence
were all cloned into the pRRL-SIN-bPRE4*
backbone under the human PGK promoter
38. Human TPco was also cloned under
the SF promoter (Figure 1A). Third-generation
packaging plasmids pMDL-g/pRRE, pRSV.
REV and envelop plasmid pMD2-VSVg wereused39. LV vectors were generated by calciumphosphate precipitation of HEK293T cells40.
48 hours after transfection, lentiviral vectors
were concentrated by ultracentrifugation
at 20, 000 rpm for 2 h at 4°C and the pellets
resuspended in PBS. LV vector titration was
performed on HeLa cells. After 72 hours, cells
were analyzed on an LSR-II flow cytometerand FACS diva software to calculate the titersof a GFP containing batch. Titers of TP, TPcocontaining vectors were determined by qPCR.

HSC transduction, and primary andsecondary transplantation
The generation of Tymp Upp1 (KO) andTymp+/+Upp1+/+wild type (WT) mice on
a C57BL/6J genetic background have been
described before22. The animals were bredin the Erasmus MC Experimental Animal
Center (Rotterdam, The Netherlands).
During the experiments, mice were kept infilter top cages, fed with autoclaved water
-/-

86

-/-

and irradiated chow ad libitum. The animal
experiments were reviewed and approvedby an ethical committee of Erasmus MC,
Rotterdam in accordance with legislation in
the Netherlands.
Bone marrow cells were harvested
aseptically from femurs and tibiae of 4 to 19
week-old male mice by flushing with PBS,
followed by lineage depletion (Lin-) according
to manufacturer recommendations (BDBiosciences). Lin- cells were cultured overnightin the presence of the LV vector constructs at
a multiplicity of infection (MOI) of 10 or 3 inserum-free modified Dulbecco’s medium with
supplements41 and stimulated with murine
stem cell factor (SCF, 100ng/mL), human
FMS-like tyrosine kinase 3 ligand (Flt3-L, 50
ng/mL), murine thrombopoietin (TPO, 20
ng/mL). Subsequently, lin- transduced cells(5×105) were injected into the tail vein of 4 to
17 weeks of age recipient KO or WT female
mice, subjected to 6 Gy irradiation twentyfour hours prior to transplantation. For
the secondary transplantation, similarly eachof two 5 to 14 week aged KO female recipientswere transplanted with 2×105 enriched
Lin- cells from viable cryopreserved bonemarrow of a single primary recipient mouse(Figure 1B).

Sample collection, analysis andtermination of experiments
During the course of experiments, primaryand secondary recipient mice were monitored
carefully for any signs of discomfort. Body
weights and leukocyte phenotypes were
measured monthly up to six months posttransplantation in some primary recipients.
Complete blood cell counts (CBCs)

HSCGT in a mouse model of MNGIE

and leukocyte phenotypes of secondary
recipients were monitored three months
after transplantation. When applicable, bloodand urine samples for biochemical analysis
were collected. Mice were removed fromexperiments if they appeared ill, had severe
body weight loss of >15%, had changes inbehavior, or for other reasons, for example
malocclusion or fight wounds. The following
analyses were performed at experimental
end point or at death when possible. EDTA
peripheral blood and urine samples werecollected on ice and stored at -80°C forbiochemical analysis. Mice were sacrificed
by inhalation of a CO2/O2 mixture andwere trans-cordially perfused with PBS.
The following was performed: assessment ofbody weights, physical appearance, presenceof abnormality such as pale feet, rectal
prolapse, injuries, lumps (tumors, cysts),visual examination of major organs (heart,
lung, brain, liver, kidney, gastrointestinal
tract, spleen, thymus, lymph nodes),documentation of organomegaly (spleen,
thymus, lymph nodes) and processing ofsamples from lesions and enlarged organs
for H&E staining/ IHC. Tissue samples were
snap-frozen in liquid nitrogen and stored at-80°C for biochemical analysis. CBCs were
measured using automated equipment (scil
Vet ABC Hematology Analyzer, Gurnee, IL).
Leukocytes, spleen cells and fractions of totalbone marrow were used for FACS analysis. In
some experiments, bone marrow cells were
cultured to assess progenitor cell content bycolony assays. Aliquots of bone marrow cells
were stored at -80°C for molecular analysis
as described below and the remainder ofthe bone marrow cells was cryopreserved
for retransplantation. For testing the riskof insertional oncogenesis, hematologicalaberrations were based on abnormal FACS
phenotype of at least one of the hematopoietic
tissues BM, spleen or peripheral blood and/or white blood cell counts (WBC) (further
analyzed for leukemia in case WBC > 25.0 x
103 cells/µl) and /or enlarged spleen, thymus
or lymph nodes. The hematological aberration
was considered a potential oncogenic clone if
both donor cells and VCN were prominent in
the recipient’s BM sample.

Flow cytometry
Erythrocytes

wereremoved

with

4

lysis

buffer (0.15M NH4Cl, 0.1M EDTA) andleukocytes, bone marrow and spleen cells
were washed with Hank’s balanced salt
solution (Invitrogen) containing 0.5% (wt/vol) bovine serum albumin and 0.05% (wt/vol) sodium azide (HBN) followed by 30
min incubation at 4ºC in HBN containing
2% heat-inactivated normal mouse serum
and antibodies against CD45.2, CD3, CD19,
CD11b , directly conjugated respectively to
APC-Cy7, APC, PE, PerCP-Cy5.5; or, against
CD8, CD45R/B220, Gr-1, c-Kit or CD4, IgD,
CD19, Sca-1 or CD3, IgM, CD11b, directly
conjugated respectively to APC, PE or PerCP(Cy5.5) (BD biosciences and Biolegend, UK,
London). Cells were washed and measured ona Canto or LSR-II flow cytometer (BD).

Progenitor cell assay
To detect colony forming unit granulocyte
monocyte (CFU-GM), 5×104 total bonemarrow cells in 10% of the total volume wereseeded in semisolid methylcellulose 80% of
the total volume (MethoCult M3231, Stemcell technology, France) with 10% of the total
volume IMDM supplemented with rm-Stemcell factor (100 ng/ml), rm-Interleukin3 (3087Chapter 4

ng/ml) and rm-GM-CSF (30 ng/ml) (R&D
systems). Cultures were incubated at 37 °C in
a humidified atmosphere containing 5% CO2
and colonies were scored seven days later
under an inverted microscope.

Biochemical analysis of TP enzyme
activity and nucleoside levels
TP enzyme activity in blood cells, tissue
samples and plasma d-Thd and d-Urd levels
were measured by liquid chromatography(UPLC-UV), while tissue d-Thd andd-Urd levels were measured by liquid
chromatography–massspectrometry(HPLC-MS) as described before19, 21. Urinary
Thd and d-Urd were measured by HPLC42.
Urine samples were thawed to room
temperature by incubation at 37Cº for 30 min
prior to measurement of concentrations. One
volume of sample plus 1 volume of internal
standard solution was diluted with 1 volumeof eluent A (30 µmM ammonium acetate,
1% Methanol, pH 4.80). The samples wereincubated at 60 ºC for 30 min, followedby centrifugation on filter units at room
temperature at 13.000 g for 20 min. 20 µl of
filtrates were then injected into HPLC system(Shimadzu, LC20 series with a binary pumpand Photodiode array detector), equippedwith an Alltima C18 5µ, 250 mm x 4.6 mmcolumn and Alltima C18 5µ guard column.
Chromatography was performed as described
before42. Concentrations were calculated bycomparison of the Thd and d-Urd peak areas
in samples to the corresponding peak areasof standards using caffeïne as an internal
standard to correct for injection volume.
Creatinine concentrations of the samples
were measured in the same runs.

88

Q-PCR and western blotting
KO Lin- cells were transduced by PGK-TP,
PGK-TPco or SF-TPco (MOI 10) and cultured
in vitro for seven days. Total RNA wasextracted and 1 µg was reverse transcribed
into cDNA (RNeasy MicroKit and QuantiTect
Reverse Transcription Kit, Qiagen GmbH,
Hilden, Germany) following manufacturer’s
instructions. Genomic DNA isolation fromboth cultured Lin- cells and bone marrowcells was performed by using NucleoSpin
Tissue kits (Bioké, Leiden, The Netherlands)
following manufacturer’s protocol. RNA
and DNA concentration and purity were
determined by using Nanodrop (Nano Drop
1000Spectrophotometer). Template cDNA
and 100ng template genomic DNA were
subject to qPCR by using SYBR Green PCR
master mix and the primers described before26,
43, 44and summarized in Table S4 (Applied
Biosystems, Foster City, CA; Eurogentec,
Maastricht, The Netherlands). The PCR
reactions were carried out in the ABI7900,
Taqman machine and results were analyzed
with SDS2.2.2 software (Applied Biosystems).
The average vector copy per cell were
calculated by comparing the cycle thresholdvalues obtained against a standard curve
obtained from mouse 3T3 or Hela cells,
and to calculate Y chromosome chimerism
a standard curve is obtained from male micebone marrow samples.
Western blotting was performed by using
Novex midi gel system for electrophoresis andblotting (Invitrogen, Carsland, CA) following
manual’s instructions. Cultured Lin- cells werelysed by sonication, protein concentrations
were determined by using BCA protein
assay kit (Thermo Fisher) and a total of 15µg proteins were separated by 4-12% Bis-Tris
SDS– PAGE, transferred to nitrocellulose andHSCGT in a mouse model of MNGIEPVDF membranes and blotted with primaryand secondary antibodies (summarized inTable S5) and finally signals were detectedwith enhanced chemiluminescent substrates
detection kit (Thermo Fisher). Image J
software was used to quantify protein levels.

Vector integration sites
In order to identify vector integration sites
vector LTR-genome junctions were amplified
by LAM-PCR as previously described45, 46.
Briefly,

after

linearbiotinylated

amplification

LTR-specificwithprimers,

amplification products were purified using
streptavidin magnetic beads. The following
steps

includedcomplementarystrandsynthesis, parallel digestion with 3 different
restriction enzymes (Tsp509 I, HpyCH4 IV
and Aci I), and ligation to a linker cassette.
The fragments generated were then amplified
by two additional exponential PCR steps.
LAM-PCR products were separated by gelelectrophoresis on Spreadex high resolution
gels (Elchrom Scientific), underwent a fusion
PCR for the addition of barcoded adaptors,
were pooled into libraries and Illumina
sequenced (MiSeq) as previously described47.
The resulting sequences were processed
through a customized informatics pipeline46,
and (Leonardelli et al. ASGCT 2016 abstract)which performs cleaning for quality andcollisions, mapping and annotating of IS on
the mouse Genome (Dec. 2011, GRCm38/
mm 10).

mtDNA analysis
Mitochondrial

DNA

copy

number,conformation and integrity were evaluatedin total and mitochondrial DNA extracted
from tissue samples48, 49. Briefly, mtDNA

copy number was determined by Real
time-PCR using primers and Taqman
probes for mitochondrial and NDUVF1 andthe AccuStartII PCR supermix (QuantaBio).
The respective primer sequences are
summarized in Table S4. To quantify
the levels of existing mtDNA strand breaks,
mitochondrial DNA and free 3’ ends werelabeled in vitro using radioactive incorporationof dCTP by terminal deoxynucleotide
transferase. 0.5 mg of purified mtDNA wasincubated with 5 uCi dCTP (3000 Ci/mmol)and 7.5 U TdT in 30 ml 1x TdT buffer at 37°C
for 30 min and separated over a 1% TBE/
agarose gel containing 0.1 mg/ml ethidium
bromide. Equal loading was confirmed by UV
visualization, the gel was Southern blotted
onto Hybond-XL membrane and the intensityof each lane was quantified by phosphor
imaging (Molecular Imager FX, BioRad).
The abundance of replication intermediates
was visualized using Brewer/Fangman 2D
neutral/neutral agarose electrophoresis.
MtDNA deletions were detected by longrange PCR as previously described50 andvisualized by Phosporimager.

4

Magnetic resonance imaging (MRI)
Imaging was performed on a 7.0 T dedicatedanimal scanner (Discovery MR901, Agilent
Technologies/GE
Healthcare)using
a 4-channel surface receiver coil (Rapid MR
International, Ohio, USA) and a 72 mm
transmit body coil. Single shot fast spinecho (SSFSE) images were acquired withsettings: TR/TE = 1600/8 ms, FOV = 5cm,
voxel size 0.2x0.2x1 mm. T2 images wereadditionally acquired using a fast pin echo
sequence for anatomical reference, settings:
TR/TE = 2500/25 ms, FOV = 5cm, voxel size
0.2x0.2x1 mm.
89

Chapter 4

Image analysis: The SSFSE is very sensitivefor high liquid content and therefore, byusing these images, the signal intensity (SI)of voxels with high liquid content is muchhigher than that of other tissue. This allowed
us to determine the fraction of edematic tissue
by selecting voxels through thresholding
after segmentation of the whole brain.
The threshold was determined by quantifying
normal tissue values by determininga reference region of interest (ROI) within
normal brain tissue. This ROI was manuallydrawn in a slice of brain without hyperintense areas. Voxels with SI of 4 standard
deviations above the reference ROI SI wereconsidered to have high liquid content (4 sd =99.9% confidence interval). The analysis was
performed using in-house developed software
based on MATLAB (MathWorks, Natick, MA,
USA).

Immunohistochemistry (IHC)
Mice were euthanized by inhalation of a CO2/O2 mixture and transcardially perfused with
PBS to remove blood. Brains were extracted
and immersion-fixed in 4% paraformaldehyde
for 24hr, paraffin-embedded and cut
longitudinally to obtain 6 µm-thick sections.
Staining was performed as previously
described31. Antigen retrieval was performedby treatment with 0.01M citrate buffer (pH6.00) in a steamer, and endogenous peroxidase
blocked with 0.3% H2O2 in methanol. Sections
were blocked in 5% normal goat serum thenincubated overnight at 4°C with the primaryand secondary antibodies summarized inTable S5. Immunoreactivity was detectedwith 3-3’-diaminobenzidine as chromogen.
Sections were finally counterstained withhematoxylin and visualized under a Leica
DM6000B microscope. Omitting the primary
antibodies yielded no significant staining.
Image series from brain sections stainedwith H&E or against protein proteolipid
protein (PLP) or the glial fibrillary acidic
protein (GFAP) were obtained with a ×40
objective. Quantification of vacuoles andastrocytes morphology was performed blindto the genotype using ImageJ. The percentageof cross-sectioned area occupied by vacuoles
was quantified in the cerebellar and callosalwhite matter. The thickness of perivascularastrocytic cell processes at the maximal width
was measured.51

Statistical analysis
Significance of differences between groups
was determined by two-tailed Mann-Whitney
U test. Data was graphed and analyzed byGraph Pad-Prism5 software (version 5.03).

| |Acknowledgments
The authors acknowledge the financial support
for this study by Join4energy, Ride4Kids,
the Sophia Foundation (SSW0645), Stichting
NeMo and the Spanish Instituto de Salud
Carlos III and FEDER funds (grant 15/000465
to RM), in the context of funding provided
by the European Commission’s 5th, 6th and7th Framework Programs, Contracts QLK3CT-2001-00427-INHERINET,
LSHB-CT90

2004-005242-CONSERT,LSHB-CT-200619038-Magselectofection, Grant Agreement222878-PERSIST and Grant agreement261387 CELL-PID, and by the Netherlands
HealthResearchandDevelopment
Organization ZonMw (Translational Gene
Therapy program projects 43100016 and
43400010).

HSCGT in a mouse model of MNGIE

We thank Dr. Michio Hirano (Dept. of
Neurology, Columbia University Medical
Center, New York, United States) for
providing the murine model, Louis Boon
(Epirus
Biopharmaceuticals,
Utrecht,
The Netherlands) for kindly providing
anti-B220 antibody, Prof. Peter A.E. Sillevis
Smitt (Dept. of Neurology, Erasmus MC,
Rotterdam, The Netherlands), Pier.G.
Mastroberardino and Chiara Milanese (Dept.

of Molecular Genetics, Erasmus MC,), Kees
Schoonderwoerd (Dept. of Clinical Genetics,
Erasmus MC) and Jeroen de Vrij (Dept. of
Neurosurgery, Erasmus MC) for valuable
discussions, Lidia Hussaarts (Dept. of Clinical
Genetics, Erasmus MC) for technical support,
King Lam (Dept. of Pathology, Erasmus MC)
for pathology evaluation and F. Dionisio
and A. Aiuti from HSR-TIGET, Milan forthe support to the integration site analysis.

| |Conflict of interest statement

4

The authors declare no conflict of interest.

| |Author contributions
Conceptualization, R.Y., N.P., M.S., I.F.M.,
G.W.; Investigation, R.Y., R.C., J.T.T., M.B.,
J.H., E.B., S.G., E.H.J., R.V.; Formal analysis,
R.Y., R.C., J.T.T., M.B., N.P., J.H., M.H., E.B.,
S.G., E.H.J., L.L.; Writing – Original Draft,

R.Y.; Writing – Review & Editing, R.Y., N.P.
I.F.M. and G.W.; Funding Acquisition, I.F.M,
G.W.; Resources, L.B., M.B.,G.R., R.M.;
Supervision, N.P., I.F.M.

91

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495

Chapter 4| |Supplementary information

Supplementary figure S1. Biochemical correction in transplanted MNGIE mice. (A)Quantifications of deoxyuridine (d-Urd) in urine, plasma of blood and brain tissues 8-11 months
after transplantation of 5×105 LV transduced Lin- cells (MOI 10, 3), n= 4-11 mice /group and (B) inintestines 11 months after transplantation of 5´105 LV transduced Lin- cells (MOI10), n= 3-9 mice /group.(C) Biochemical correction in skeletal muscle and liver tissues 11 months after transplantationof 5×105Lin- (MOI 10), n= 3-9 mice /group. The horizontal line represents the median. *P <0.05,**P <0.01 and ***P <0.001, n.s. = not significant.

96

HSCGT in a mouse model of MNGIE

Supplementary figure S2. Severe body weight loss in primary recipients of SF-TPco. PGK-TP,
PGK-TPco and SF-TPco recipients and untreated KO mice were divided into two groups based on
severe body weight loss at the time of death (>15% weight loss occurred over a time span of 1 to 2
months, SF-TPco, median body weight 15.7g ( range, 14.3-17, n= 6) and a recipient of PGK-TP (14g) and the other group are mice which gained or did not remarkably lose weight until termination(n= 39), plotted on the y-axis the survival of animals in these 2 groups. The horizontal line represents
the median, ***P <0.0001.

4

A

B

Supplementary figure S3. LV integration site analysis. (A) Schematic representation of invitro and in vivo experiments for identification of LV integration sites. Bone marrow Lin- cells weretransduced (MOI 10 ) overnight and were either cultured for seven day, or transplanted into KO or
WT recipient mice. a Integration site analysis was performed on the in vitro cultured Lin- cells and BM
of primary recipients. The vector copy number (VCN/ cell) measured in Lin- cells, n= 2 experiments,
and VCN/ cell and BM chimerism measured in bone marrow cells, n= 2 -5 mice/group, is illustratedin (B). The horizontal line represents the median.

97

Chapter 4

Supplementary figure S4. Characterization of LV vector positive B-cell lymphoma. (A) FACS
blot of BM cells stained for B220+ and demonstration of the size (FSC) and granularity (SSC) of
the B220+GFP+ population. (B) Histology and immunohistochemistry for large B-cell lymphoma
infiltration in liver. H&E and B220 staining (200x) (C) Overview of the PGK-TP-GFP insertion into
the third intron of Zfp207 gene as identified by LAM-PCR and sequencing analysis of BM.

98

6.5

(2.7-11.4)
4.4(3-5.7)
6.2(5.2-7.2)
5.1(3.9-9.3)
4(1.9-5.4)
5.8

(3-7)

3.7

(3-4)

KO

(n=7)
WT

(n=6)
PGK-TP(n=5)
PGK-TPco(n=6)
SF-TPco(n=4)
PGK-GFP(KO-KO)(n=4)
PGK-GFP(WT-WT)(8-9.4)8.1(9.4-9.9)(3-9.2)9.6(3.4-9.5)
5(8-10.2)
7.7(8.5-10.6)
8.9(9.3-10.6)
9.1

9.8

RBC
(x106/μl)(7.2-9.2)

7.8(8.6-9)(3-8.3)
8.7

(4-9)
4.8

(7.6-9.1)
7.2(7.8-9.6)
8.1(8.3-9.6)
8.4

9.1

HGB
(x105)(0.41-0.48)

0.41(0.46-0.5)(0.1-0.4)
0.48(0.21-0.48)
0.26(0.4-0.5)
0.39(0.4-0.5)
0.46(0.45-0.52)
0.46

0.5

HCT
(x105)(590-793)

665(609-1236)(773-1198)
1086(240-1531)
1145(931-1705)
861(473-1489)
1277(974-1529)
901

1196

PLT
(x109/l)(51-52)

51.5(48-54)(49-54)50(50-63)
52(49-52)
51.5(48-52)50(48-51)
50

50

MCV

MCHC
18.4
(17.80-18.90)
18.2(17.9-19.1)
18.2(17.1-19)
18.3(17.6-18.7)
18(17.3-18.5)
18(17.2-18.4)
18.8(17-19)MCH
0.93(0.89-0.95)
0.91(0.89-0.98)
0.93(0.88-0.95)
0.96(0.90-1.1)
0.94(0.9-0.96)
0.90(0.88-0.92)
0.97(0.88-0.97)a

WBC: white blood cells; RBC, red blood cells; HGB, hemoglobin; HCT, Hematocrit ;PLT, platelets; MCV, mean cell volume; MCH, mean cell hemoglobin; MCHC,
Mean cellular hemoglobin concentration. Data represent median (range)

(n=4)

WBC
(x103/μl)

LV vector

Supplementary table S1. Complete blood counts (CBC) of gene therapy treated primary recipient mice and control groupsa

HSCGT in a mouse model of MNGIE

4

99

100
(0-0.1)

0.1

Plasmad-Urd(μM)(133-501)240(0.2-1.6)

0.6

Brain TP activity
Brain d-Thd(nmoles
thymine/h/mg(pmoles/mg
protein)protein)(0.1-3)

1.6

Brain d-Urd(pmoles/mg
protein )98

BM cellchimerism(%)(0.3-11) (75-100)

1.8

VCN/
cell

Quantification of thymidine phosphorylase (TP) enzyme activity, thymidine (d-Thd) and deoxyuridine (d-Urd) 11 months after transplantation of 5×105 LV-SF-TPcotransduced Lin- cells (MOI 10). Data represent median (range), n= 2- 7 mice.

(0-0.1)

0

0

0

406

(351-438)

Urnie d-Urd Plasma(mmol/mol d-Thd
creatinine) ( μM)

Blood TP activity(nmoles
Urine Thdthymine/h/mg(mmol/mol
protein)creatinine)

Supplementary table S2. Biochemical and molecular data of LV-SF-TPco treatment group.

Chapter 4

2.8(0.77-100)0.22(0.04-16)

12

12

20

6

PGK-TP

PGK-TPco 6

SF-TPco1(0.1-47.2)(0.0-0.5)(0.10-100)

0.01

2

(0.01-4.4)

30.4

0.12

0.52

0

0

0

1 (diarrhea, wasting)(n=2; 57%, 35%)

Gr-1+CD11b+ Myeloid cells

CD4+CD8+ T cells (n=3; 40%,68%, 7%)2 (wasting)(n=1; 64%, WBC= 57×103/μl)

1 (passive)
1 (found dead)2 (wasting)1 (reactive pathology/ hepatic
steatosis observed at autopsy)

1 (wasting)

2 (malocclusion, wasting)

3 (wasting)

1 (malocclusion, wasting)1 (found dead)

B220+ B cell- leukemia

6(85%,WBC=53×103/μl)

B220+ B cell- leukemia

(52%)
1

CD4+CD8+ T cells

B220+GFP+ B-cell
lymphoma (83%, 85%)01

2

5 (wasting)5 (found dead)Number of prematurely
dead mice or mice sacrificed
with high discomfort scores e

Mice were transplanted with LV transduced 5×105 BM Lin- cells ( MOI 10 or 12) and followed for 8-11 months after transplantation. Two secondary recipients were transplanted with
2×105 BM Lin- cells isolated from one primary recipient mouse. a, b Vector copy number per cell (VCN/cell) and bone marrow chimerism of all the secondary recipients in each treatmentgroup are presented as median (range), n=3-16/ group. c, d Hematological aberrations and clonal expansion of transduced cells were assessed based on altered FACS phenotypes and/or
elevated WBCs ( > 25.0 x 103 cells/µl= leukemia), and /or enlarged spleen, thymus or lymph nodes of mice with high discomfort scores. The percentage of the abnormal BM phenotypiccells are indicated between parenthesis. The hematological aberration is vector-positive if both donor cells and VCN were prominent in the recipient’s BM. e The right column includes
mice that were found dead or mice sacrificed prematurely during the experiment due to high discomfort scores and hematological aberrations could not be identified (details are specified
between parenthesis). f PGK-GFP group include recipient mice of KO or WT Lin- cells transduced by PGK-GFP.

10(0.21-0.86) (3-67.3)

12

6

B220+ B cells (86%)

PGK-TPGFP

0

1(0.03-0.12) (0.16-0.4)

0.25

32

PGK-GFP f 16

0.09

Number of non-vector-positive
hematological aberrations d

Number of
Number Number ofBM cell
vector-positiveof donor secondary
chimerism hematological
LV vector micerecipients VCN/cell a (%) b
aberrations c

Supplementary table S3. Incidence of hematological abnormalities in secondary recipients of LV transduced cells

HSCGT in a mouse model of MNGIE

4

101

102

Mouse GAPDH
WPRE
Mouse mtDNA1978F, 2086R
Mouse NDUFV1Mouse mtDNA probe
Mouse NDUFV1 probe

CTGGAAGGGCTAATTCACTCCATCGGAGGGCTAAAGTGTCAC

HIV-1 (U3-Psi)
SRY

GGTTTCCCTTTCGCTTTCAGTGGCATGTGGGTTCCTGTCC

Reverse primer(5’>3’)
Employed for

Detection of integrated viral copies (VCN/cell)
Detection of male chromosome (Donor cellchimerism)
ACGGCAAATTCAACGGCACAG
ACACCAGTAGACTCCACGACATAC Internal control
GAGGAGTTGTGGCCCGTTGTTGACAGGTGGTGGCAATGCCWPRE mRNA expression
TGCCTGCCCAGTGACTAAGGACCCTCGTTTAGCCGTTCA
Quantify mtDNA copynumber
ATCCSAGGATCCCACAGAGCT
CCTTTCCAGCAGATGTGGGTFAM-TGACCGTGCAAAGGTAGCAT-MGB
VIC-GAGCCTTAGGGAAGAGGCAG-MGB

Forward primer(5’>3’)

Abbreviation

Supplementary table S4. Primers used in quantitative PCR (qPCR)

Chapter 4

HSCGT in a mouse model of MNGIE
Supplementary table S5. Antibodies used in this study
Anti-

Clone

Host organism Vendor

Human TP,P-GF.44C
55kDa
GAPDH, 36 kDa 6C5

Mouse

Mouse IgG H&L
Mouse PLP

Polyclonal
Plpc1

Goat
Mouse

Mouse MBP

Mouse

Mouse GFAP
Mouse B220

Not specified
by vendor
Polyclonal
RA3.6B2

Mouse IgG
Rat IgG H&L

Polyclonal
Polyclonal

Goat
Rabbit

Mouse

Chicken
Rat

Dilution

Calbiochem-Merck, 1:250
GF40
Applied Biosystems, 1:250
AM4300WB*/IHC*
WB

Abcam, Ab97023
AbD Serotec,
MCA839G
Millipore, MAB387

1:10001:3000

WB
WB
IHC

1:50

IHC

Millipore, Ab5541
EpirusBiopharmaceuticals
Dako, K4000
Vector labs,
BA-4000

1:1000
1:50

IHC
IHC

1:1000
1:150

IHC
IHC

4

*WB, Western blot. *IHC, Immunohistochemistry.

103

Transplantation, gene therapy and intestinal
pathology in MNGIE patients and miceRana Yadak1,2, Max V. Boot3, Niek P. van Til2,4,Dominique Cazals-Hatem5, Armin Finkenstedt6, Elly Bogaerts7,
Irenaeus. F. de Coo*1,8, Marianna Bugiani*3
1
Department of Neurology, Erasmus University Medical Center, Rotterdam, The Netherlands;
Department of Hematology, Erasmus University Medical Center, Rotterdam, The Netherlands;
3
Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands; 4 Laboratory ofTranslational Immunology, University Medical Center Utrecht, Utrecht, The Netherlands; 5Departmentof Pathology, Beaujon Hospital, Paris, France; 6 Department of Medicine I, Medical University of
Innsbruck, Innsbruck, Austria; 7Department of Clinical Genetics, Erasmus University Medical Center,
Rotterdam, The Netherlands; 8Department of Clinical Genetics, Maastricht University Medical Center,
Maastricht, The Netherlands
2(Submitted)

5Chapter 5| |Abstract
Gastrointestinal complications are the main cause of death in patients with mitochondrial
neurogastrointestinal encephalomyopathy (MNGIE). Available treatments often restore
biochemical homeostasis, but fail to cure gastrointestinal symptoms. We evaluated small
intestine histology of an untreated MNGIE patient and two recipients of hematopoietic stem
cells. Additionally, we evaluated the intestinal pathology in a mouse model of MNGIE treatedwith hematopoietic stem cell gene therapy. Our data confirm that MNGIE presents with muscle
atrophy and loss of Cajal cells and CD117/c-kit immunoreactivity in the small intestine. We also
show that hematopoietic stem cell transplantation does not benefit human intestinal pathology
on short-term.

106Intestinal pathology in MNGIE| |Introduction
Mitochondrial
neurogastrointestinal
encephalomyopathy (MNGIE) is a rare
inherited metabolic disorder caused by lossof-function mutations in the nuclear gene
TYMP leading to thymidine (Thd) anddeoxyuridine(d-Urd)accumulation.1
Alongside classic neurological signs (external
ophtalmoplegia, leukoencephalopathy andsensorimotor peripheral neuropathy), chronic
intestinal pseudo-obstruction (CIPO) is
reported in almost all MNGIE patients andoccurs at onset in 45-67% of cases.2, 3 Other
gastrointestinal symptoms include earlysatiety, nausea, dysphagia, post-prandial
emesis, abdominal pain and/or distention
and diarrhea.4
Allogeneic
hematopoietic
stem
cell transplantation (HSCT) corrects

the biochemical metabolic imbalance as
donor-derived

leucocytes

andplatelets

are rich in thymidine phosphorylase.4 It iseffective to relieve CIPO in few reported
MNGIE cases5 although malnutrition often
persists and most cases rely on nutritional
support.6 Neurogenic and myogenic changes
and alterations of the interstitial Cajal cells,
the gut pacemakers, were reported in MNGIE
patients.7-10 Restoration of gastrointestinal
integrity by available treatments however
has not yet been addressed. Also, whether
small intestine pathology is recapitulated in5

the mouse model of MNGIE is not known.11,12
In this study, we also evaluated the effects oftreatment on small intestinal pathology of
MNGIE patients and mice.

| |Subjects and Methods
MNGIE patients and controls

TYMP sequence13, and the third-generation14

Table 1 reports the demographic data ofthree MNGIE patients and three controls.
Patients were diagnosed based on clinical,
biochemical, and molecular features.1 One
patient was untreated, two received HSCT.
Written informed consent was obtained forall subjects.

packaging and envelop plasmids were used
to generate LV-PGK-TP vector particles, bycalcium-phosphate precipitation of HEK293T
cells.15 Titration was performed on HeLa
cells and titers determined by quantitative
polymerase chain reaction (qPCR). Donor KO
male bone marrow lineage-depleted (Lin-) cells(BD Biosciences) were transduced overnight

MNGIE micewith the lentiviral vector at a multiplicity

Tymp Upp1 (KO) and Tymp Upp1 wild
type (WT) mice11 were bred in filter top cages
and fed ad libitum with autoclaved waterand irradiated chow. Animal experiments
were approved by the ethical committeeof the Erasmus University Medical Center,
Rotterdam, in accordance with Dutch
legislation.
pRRL.PGK.TYMP.bPRE4*.SIN
self-inactivating lentiviral transfer plasmid
containing the human PGK promoter driving
-/-

-/-

+/+

+/+

of infection 10 in serum-free modified
Dulbecco’s

mediumwithsupplements,16

conditioned with murine stem cell factor,
human Flt3-L, and murine thrombopoietin.
Five to 10-week-old recipient KO female micereceived 6Gy total body irradiation 24 hours
prior to tail vein injection of 0.5×106 Lintransduced cells. Bone marrow genomic DNA(Bioké, Leiden, The Netherlands) was used as
template for qPCR using primers and SYBR
107

108

18 y
19 y
Diarrhea, vomiting, weight loss,
abdominal pain, liver steatosis
Ptosis, peripheral neuropathy,
neurogenic bladder,leukoencephalopathy, lactic
acidosis, hypertriglyceridemia
TP deficiency- NoneAlive (6 y)Age of onset
Age at diagnosis
GI symptoms

Extra-GI symptoms

Treatment of
MNGIE (age)

Follow-up

MNGIE-3

c.866A>C in TYMP
Allogeneic HSCT
(17 y)
GVHD, sepsis; died 6months after treatmentc.866A>C in TYMP
Allogeneic HSCT
(34 y)
Multi-organ failure; died 18
days after treatment

10 y
17 y
Diarrhea, weight loss,
abdominal pain
Retinopathy,
peripheral neuropathy,
leukoencephalopathy

Urinary d-Urd,

23 y
32 y
Diarrhea, weight loss, liver
steatosis
External ophtalmoplegia,
peripheral neuropathy,
leukoencephalopathy

MNGIE-220
Control-1

NA-

Pancreatitis

NA

NA
41 y
NA

Control-2

NA

-

IOPN

NA

NA
46 y
NA

Control-3

NA

-

GIST

NA

NA
28 y
NA

GI, gastrointestinal. NA, not available. TP, thymidine phosphorylase enzyme. d-Urd, deoxyuridine. TYMP, thymidine phosphorylase. IOPN, intra-ductal oncocytic
papillary neoplasm. GIST, gastrointestinal stromal tumor. HSCT, hematopoietic stem cell transplantation. GVHD, graft-versus-host disease.

Diagnosis

MNGIE-1

Patient

Table 1. Clinical and molecular data of MNGIE patients and controls

Chapter 5

Intestinal pathology in MNGIEGreen PCR master mix (Applied Biosystems,
Foster City, CA; Eurogentec, Maastricht,
The Netherlands). PCR reactions were carriedout in the ABI7900, Taqman machine, andanalysis performed with SDS2.2.2 software(Applied Biosystems). The Cycle thresholdvalues were compared against a standard
curve obtained from mouse 3T3 to calculate
average vector copy per cell, or from malemice bone marrow to calculate Y chromosome
chimerism.
High performance liquid chromatography(Shimadzu, LC20 series with a binary pumpand Photodiode array detector)17 equippedwith an Alltima C18 5µ, 250 mm x 4.6 mmcolumn and Alltima C18 5µ guard column
was used to measure urinary Thd and d-Urd.

and Phosphotungstic acid-hematoxylin andimmunostained as described18 against smoothmuscle actin (SMA, Dako, 1:200), CD117/ckit (Dako, 1:50), calretinin (Dako, 1:200),
CD3 (Dako, 1:250), CD8 (Dako, 1:50) andglial fibrillary acidic protein (GFAP, Dako,
1:300). Immunoreactivity was detected using
3,3’-Diaminobenzidin or Liquid Permanent
Red as chromogen. Pictures were taken witha Leica DM3000 microscope. Muscle thickness
was measured on transversally cut intestinal
sections (N=40 for mice, N>25 for humans).
Quantification of muscle wall thickness andganglion cell density was performed blind to5

the genotype with ImageJ.

Statistical analysis

Pathological analysis

Data were analyzed with Graph Pad-Prism5

Formalin-fixed paraffin-embedded 5-μmthick small intestine tissue sections wereroutinely stained for Hematoxylin-Eosin(version 5.03). Significance of differences
between groups was determined by two-tailed
Mann-Whitney U test (P<0.05).

| |Results
Small intestine pathology in MNGIE
patients

Small intestine pathology in MNGIE
mice

Microscopic analysis (Fig 1) revealed
a preserved layer composition of the small
intestine in all MNGIE patients with novillous atrophy or significant inflammation.
In MNGIE patients, however, the externallayer of the tunica muscularis propria
was fibrotic and thinner than in controls,suggesting muscle atrophy. The submucosal
plexus appeared normal. In the myentericplexus, no significant loss or morphologic
abnormalities of ganglion cells and entericglial cells were observed. Cajal cells, however,identified by CD117/c-kit immunoreactivity,
were completely lost in all MNGIE patients.

We investigated small intestine histopathology
in MNGIE mice using 2 (young) and 12month-old animals (old) to also check forsigns of progression (Fig 2). HematoxylinEosin staining revealed significant atrophyof the tunica muscularis propria and loss ofmyenteric ganglion cells in old KO compared
to young Tymp-/-Upp1-/- and old WT mice.
Unfortunately, CD117/c-kit immunostaining
was unsuccessful.
We then assessed the effects of treatment
on the MNGIE phenotype (Fig 2).
Following hematopoietic stem cell genetherapy (HCSGT), vector copies number
109

Chapter 5

per cell, engraftment levels and urinary
nucleosides concentrations indicated efficient
hematological reconstitution and biochemicalcorrection. Histopathology showed recoveryof tunica muscularis propria thickness in oldtreated mice compared to Tymp-/-Upp1-/-mice;
however, no significant changes were observedin the number of myenteric ganglion cells.

Figure 1. Small intestinal histopathology in MNGIE patients. (A, B) Hematoxylin-Eosin(H&E) stains of the small intestine of control subjects (A) and MNGIE patients (B) show normallylayered organization of the wall in both groups (M: tunica mucosa; S: tunica submucosa; MP:tunica muscularis propria; S: tunica serosa). (C, D) Compared to controls (C), phosphotungstic acidhematoxylin (PTAH) stains of MNGIE small intestines (D) show thinning of the external layer of
the tunica muscularis propria (blue, arrows). (E) Quantification demonstrates muscle wall atrophy inthe three MNGIE patients compared to two controls. One control was omitted because the tunica
muscularis was incompletely present. (F, G) Immunostain against calretinin shows presenceof ganglion cells in the submucosal Meissner plexus of controls (F) and MNGIE patients (G). (H)Quantification of myenteric ganglion cells shows similar cell density in MNGIE patients and controls.
(I, J) Immunostain against CD117 shows normal presence of interstitial Cajal cells around groupedmyenteric ganglion cells in controls (I), whereas Cajal cells are completely depleted in MNGIE patients(J). Small immunopositive cells in J are mast cells. (K, L) Immunostain against the glial fibrillary acidic
protein (GFAP) shows normal immunoreactivity in myenteric ganglion and enteric glia cells in MNGIE(L) as in controls (K). In both graphs bars denote the median. Original magnifications (A, B): 12.5x;(C, D): 25x; (I, J): 400x; (K, L): 200x. ***P<0.001.

110

Intestinal pathology in MNGIE

5

Figure 2. Small intestinal histopathology in MNGIE mice. (A, B) Hematoxylin-Eosin (H&E) stains of
the small intestine of young (2-month-old) control mice (A) and age-matched Tymp-/-Upp1-/- mutants(B) show normally layered organization of the intestinal wall in both groups. (C, D) H&E stains of
the small intestine show normal thickness of the tunica muscularis propria in old (12-month-old)control mice (C), whereas in age-matched Tymp-/-Upp1-/- mutants (D) the muscle wall is atrophic. (E)Bone marrow cell chimerism and vector copy number in recipients of 0.5×106 Lin- cells transduced
by LV-PGK-TP (MOI10) (n=3 mice). (F) Quantification of Thd and d-Urd in urine of untreated controls
and age-matched recipients 6 and 11 months after transplantation (n=3 mice). (G) H&E stain of
the small intestine shows recovery of normal thickness of the tunica muscularis propria in old (12month-old) Tymp-/-Upp1-/- mice 10 months after treatment. (H) Quantification confirms atrophy ofthe muscle wall in 12-month-old Tymp-/-Upp1-/- mice compared to wild-type age-matched controls.
Treatment is associated with normal thickness of the tunica muscularis propria. (I) Quantification of
the number of myenteric ganglion cell groups per tissue section shows progressive loss of ganglion
cells in Tymp-/-Upp1-/- mice, without effect of the treatment. N=2-4 mice/group; in all graphs lines
represent the median; *P<0.05, **P<0.01, ***P<0.001. Original magnification (A-D and G): 200x.

111Chapter 5| |Discussion
MNGIE is associated with gastrointestinal
symptoms, including CIPO2,3. Limited
understanding of the pathological andmolecular
mechanisms
underlying
gastrointestinal complications in MNGIE
stems from limited availability of patient tissue
and models11 that accurately recapitulate
the human gastrointestinal pathology. Our
study confirms the morphological changes
in human MNGIE small intestine, including
atrophy and fibrosis of the external layer of
the tunica muscularis propria.8 The selective
involvement of this layer has been attributed
to physiologically very low mitochondrial
DNA amounts making this compartment
selectively vulnerable to disease.8 Muscle wall
atrophy was more pronounced in transplanted
patients, possibly due to additional HSCTrelated stress. We also found complete lossof interstitial Cajal cells and networks inpatients’ small intestine. Cajal cells play roles
in orchestration of normal gastrointestinal
motility and in dysmotility disorders.19
One study previously described similarfindings in a MNGIE patient.9 Altogether,
these findings suggest that Cajal cell loss
is a cellular substrate of human MNGIE
gastrointestinal pathology.
We used Tymp-/-Upp1-/- mice, a model of
MNGIE, to investigate whether it recapitulates
the human gastrointestinal pathology.
In mutant mice, histopathology revealed
atrophy of the tunica muscularis propria
as in MNGIE patients and, in addition, lossof myenteric ganglion cells. These features
were more prominent in old Tymp-/-Upp1/compared to young animals. In Tymp-/Upp1-/- mice, we did not see presence ofCajal cell on routine Hematoxylin-Eosin

112

stained sections. Unfortunately, CD117/c-kit
immunostaining of mouse small intestine was
unsuccessful, so that we cannot conclude on
Cajal cells absence in Tymp-/-Upp1-/- mice.
However, taking our patient and mouse
data together, we can suppose a sequence of
pathological events leading to MNGIE small
intestinal disease, including loss of interstitial
Cajal cells and their networks followed byatrophy of the tunica muscularis propria andeventually loss of myenteric ganglion cells9.
The observed loss of myenteric ganglion cells
in the mice, but not in patients’ small intestine
may be attributed to inter-species differences
and the construct of the MNGIE mousemodel being knock-out for both diseaserelated enzymes. Our study describes, forthe first time, the small intestinal pathology
in MNGIE patients treated with HSCT.
Although the study is limited by the small
patient number and relatively short time
lapse between treatment and demise, HSCT
did not significantly change the trend tointestinal muscle wall atrophy and complete
loss of Cajal cells in treated compared tothe untreated patient. This suggests that
MNGIE small intestinal pathology may
not be recovered upon HSCT, which could
explain the repeatedly reported insufficient
improvement of gastrointestinal symptoms in
MNGIE patients after treatment.5
The HSCT procedure carries a highmortality rate5. Recently, HSCGT has been
explored in Tymp-/-Upp1-/- mice, providing
higher enzymatic levels compared to HSCT
and abating the risk of graft-versus-hostdisease.12 Due to intrinsic limitations of
the mouse model, i.e. lack of an apparent
clinical phenotype, only biochemical

Intestinal pathology in MNGIE

correction was shown after HSCGT. Moreover,the pathological changes in the intestine of
Tymp-/-Upp1-/- mice were never evaluated.12
Here, we show that the transplanted genemodified cells engrafted well in recipient mice,leading to clearance of systemic nucleosides.
The observed atrophy of the tunica muscularis
propria was recovered upon HSCGT, whereas
the degree of myenteric ganglion cell loss
remained unchanged. Similarly as for MNGIE
patients, recovery of ganglion cells may
take longer than our follow-up of the mice.

Alternatively, the possibility that MNGIE
permanently affects ganglion cells, including
their precursors, cannot be excluded.
In conclusion, our data suggest that
allogeneic HSCT may be insufficient tocorrect gastrointestinal pathology completely,
especially at later stages of MNGIE. As
interstitial Cajal cells and their networks playa key role in development of gastrointestinal
dysmotility,
alternative
therapeutic
approaches taking absence of these cells into
account could be required.

| |Author contributions
RY, IFdC and MBu conceptualized the study.
RY and MBo performed the experiments. RY,
MBo and MBu analyzed the data. RY and MBu

wrote the manuscript. NvT, DCH, AF, MF, EB,
IFdC reviewed and edited the manuscript.
IFdC and MBu share senior authorship.

5| |Conflicts of Interest
None.

| |Acknowledgments
We would like to acknowledge the financial
support of Join4energy, the Sophia Foundation(SSW0645) and Stichting NeMo. We
acknowledge M. Hirano for providing
the Tymp-/-Upp1-/- mice. We thank M. Doukas

and the Erasmus medical centre tissuebank for selecting and providing the tissueof the control subjects. We are gratefulto P. Sillevis Smitt for critically reading
the manuscript.

113

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12.

Torres-Torronteras J, Cabrera-Perez R, Barba
I, et al. Long-Term Restoration of Thymidine
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Torres-Torronteras J, Gomez A, Eixarch H,
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Van Acker KJ, Eyskens FJ, Verkerk RM,
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CGACTGCGGCAGTG

CTGGGATGGGAAGCCGGGAAATCAGGCATGGTGACCTGATCCTGAATGG

GGTCAGC

CCCCACC

CTTCGGG

AGTGGCCCGACTGC

GGCAGTG

CTGGGATGGGAAGCCGGGAAATCAGGCATGGTGACCTGATCCTGAATGG

GGTCAGC

CCCCACC

CTTCGGG

AGTGGCCCGACTGC

GGCAGTG

CTGGGAT

6

Hematopoietic stem cell lentiviral integration
profiles are stable across multiple species,disease phenotypes and hematopoietic
CCGAGCTCCGCCCTCCAGGCGGCCCCACCCGCCTGCCGTCCTGGGGCGCCGCCGCCCCGCCGC
cell types

GGACCGCTGTGCGCGAACCCTGAACCCTACGGTCCCGACCCGCGGGCGAGGCCGGGTACCTGG
Marshall W. Huston1, 2, Sebastiaan Horsman3, Ali Nowrouzi4,5,

TCCGGAGCAAGCGGGCGAGGGCAGCGCCCTAAGCAGGTACGGGCGGGGCTCAAGTCGCGAGGC
Guus van der Velden1, Amir. K.Varkouhi1,6, Yuedan Li1,7,Rana Yadak , Merel Stok , Fatima Aerts Kaya ,CGGGAGGCAGACACGGACGAGGGCGACACAGACACGGGACCGAGGGGCGGACACCGGAGAGAC
1
2
3
1,2

1,8

1,9

Elnaz Farahbakhshian , Irenaeus F. de Coo , Andrew Stubs ,Manfred Schmidt5, Christof von Kalle5, Peter van der Spek3,
AGGGGTCGGGACAGGAGCACGTGGCTCAGACACCGACGCCGGGAGGCCGCAGACCCCGGACGT
Gerard Wagemaker*1,9 and Niek P. van Til*1,10

ATCCCCGCAGGCCCGGAGCGATGGCAGCCTTGATGACCCCGGGAACCGGGGCCCCACCCGCGC
Department of Hematology, Erasmus University Medical Center, Rotterdam, the Netherlands, 2Current

1

affiliation: Department of Neurology, Erasmus University Medical Center, Rotterdam, the Netherlands,
CTTCTCCGGGGAAGGGAGCCAGGGACTTCCCGACCCTTCGCCAGAGCCCAAGCAGCTCCCGGA
Department of Bioinformatics, Erasmus University Medical Center, Rotterdam, the Netherlands,

3

Department of Translational Oncology, National Center for Tumor Diseases and German Cancer
CGCATGAAGCGAGACGGAGGCCGCCTGAGCGAAGCGGACATCAGGGGCTTCGTGGCCGCTGTG
4

Research Center (DKFZ), Heidelberg, Germany, 5Current affiliation: Division of Translational

RadioOncology, National Center for Tumor Diseases and German Cancer Research Center (DKFZ),GGAGCGCGCAGGGCGCACAGATCGGTGCGTGGGGAGGGTTGGGCGTTCCTGACCCCGACTGGG
Heidelberg, Germany, 6Current affiliation: Department of Ophthalmology and Visual Sciences,

University of Alberta, Edmonton, Canada, Current affiliation: Department of Radiology, Leiden
CCCGAGAGACTTTGGGTCCCTGGGGGTGCGACGGTGCCCCACTACCAGCACCGGCCCCAGGGT
7

University Medical Center, Leiden, the Netherlands, 8Current affiliation: Department of Pediatrics,
Department of Clinical Genetics, Center for Lysosomal and Metabolic Diseases, Erasmus University
Medical Center, Rotterdam, the Netherlands, 9Current affiliation: Stem Cell Research and Development
Center, Hacettepe University, Ankara, Turkey, 10Current affiliation: Laboratory of Tumor Immunology,
University Medical Center Utrecht, Utrecht, the Netherlands

CGCTGTGGGCTGCCACCCTCACGCGTACCCCCACATACCAGGGGCCATGCTGATGGCCATCCGGGCATGGATCTGGAGGAGACCTCGGTGCTGACCCAGGCCCTGGCTCAGTCGGGACAGCAGCTG

CAGAGGCCTGGCGCCAGCAGCTTGTGGACAAGCATTCCACAGGGGGTGTGGGTGACAAGGTCA(To be submitted)

CCGAGCTCCGCCCTCCAGGCGGCCCCACCCGCCTGCCGTCCTGGGGCGCCGCCGCCCCGCCGC

GGACCGCTGTGCGCGAACCCTGAACCCTACGGTCCCGACCCGCGGGCGAGGCCGGGTACCTGG

TCCGGAGCAAGCGGGCGAGGGCAGCGCCCTAAGCAGGTACGGGCGGGGCTCAAGTCGCGAGGCCGGGAGGCAGACACGGACGAGGGCGACACAGACACGGGACCGAGGGGCGGACACCGGAGAGACAGGGGTCGGGACAGGAGCACGTGGCTCAGACACCGACGCCGGGAGGCCGCAGACCCCGGACGTATCCCCGCAGGCCCGGAGCGATGGCAGCCTTGATGACCCCGGGAACCGGGGCCCCACCCGCGC

CTTCTCCGGGGAAGGGAGCCAGGGACTTCCCGACCCTTCGCCAGAGCCCAAGCAGCTCCCGGACGCATGAAGCGAGACGGAGGCCGCCTGAGCGAAGCGGACATCAGGGGCTTCGTGGCCGCTGTG

GGAGCGCGCAGGGCGCACAGATCGGTGCGTGGGGAGGGTTGGGCGTTCCTGACCCCGACTGGG

CCCGAGAGACTTTGGGTCCCTGGGGGTGCGACGGTGCCCCACTACCAGCACCGGCCCCAGGGT

CGCTGTGGGCTGCCACCCTCACGCGTACCCCCACATACCAGGGGCCATGCTGATGGCCATCCGGGCATGGATCTGGAGGAGACCTCGGTGCTGACCCAGGCCCTGGCTCAGTCGGGACAGCAGCTG

CAGAGGCCTGGCGCCAGCAGCTTGTGGACAAGCATTCCACAGGGGGTGTGGGTGACAAGGTCACCGAGCTCCGCCCTCCAGGCGGCCCCACCCGCCTGCCGTCCTGGGGCGCCGCCGCCCCGCCGC

GGACCGCTGTGCGCGAACCCTGAACCCTACGGTCCCGACCCGCGGGCGAGGCCGGGTACCTGG

TCCGGAGCAAGCGGGCGAGGGCAGCGCCCTAAGCAGGTACGGGCGGGGCTCAAGTCGCGAGGC

Chapter 6| |Abstract
The potential of ex vivo hematopoietic stem cell (HSC) gene therapy to treat monogenic inherited
disorders has been convincingly demonstrated in clinical trials. These trials also underlined
the inherent genotoxic risks of gammaretroviral vectors and the possible contribution of
the disease background. To acquire more insight on lentiviral integration patterns, human,rhesus and murine hematopoietic stem cell progenitors were transduced and expanded in vitro.
Gene corrected murine progenitor cells were subsequently transplanted into severe combined
immunodeficiency, Pompe disease, mitochondrial neurogastrointestinal encephalomyopathyor healthy mice. Vector-genome fusion sequences were pooled into an over 12,000 lentiviral
integration sites (LVIS) database. The results demonstrated consistent integration profiles among
the three species studied, and neither promoter choice nor transgene significantly influenced
the integration pattern in vivo. LVIS flanking oncogenes occurred at a frequency consistent
with random distribution. Importantly, the spleen focus forming virus promoter vectors did
not differ in integration pattern from those with cellular promoters. Genes near the detected
LVIS found in vitro and in vivo were involved in different overrepresented functional pathways,
suggesting that either the long-term repopulating HSC differ in integration pattern from actively
proliferating progenitors or a negative selection of certain integration sites occurred during
hematopoietic reconstitution in vivo.

118

Hematopoietic stem cell lentiviral integration profiles| |Introduction
Clinical hematopoietic stem cell genetherapy trials implemented since 2000 havesuccessfully reversed the disease phenotype
in a number of patients with life-threatening
inherited diseases, but also underlined
the risks associated with genomic integrationof the viral vectors used. Trials for X-linked
severecombined
immunodeficiency
adenosine
deaminase(SCID-X1)1, 2,
3, 4
deficiency (ADA-SCID) , X-linked chronic
granulomatous disease (CGD)5, WiskottAldrich syndrome (WAS)6, 7, β-thalassemia8,
andX-linkedadrenoleukodystrophy9
10metachromatic leukodystrophy all reported
that the majority of patients obtained a clear
clinical benefit to their disease phenotype.
However, in the SCID-X1 trials five patients(out of 20 treated) developed acute clonal T
cell lymphoproliferative disorders caused by
the insertional activation of proto-oncogenes
LMO2 or CCND211, 12. Four of these patientswere successfully treated with chemotherapy
without compromising the effectiveness of
their gene therapy treatment, while the fifth
patient succumbed in spite of treatment.
Two CGD patients treated with gene therapy
developed myelodysplasia due to insertional
activation of the proto-oncogene MDS1EVI1, with one patient undergoing allogeneic
hematopoietic stem cell transplantation andthe other dying of sepsis13. Seven out of ten
patients in the WAS gammaretroviral clinical
trial developed acute lymphocytic leukemia
similar to that seen in the SCID-X1 trials6, 14.
These vector-associated adverse events,
which had been observed in experimental
settings only at a very low frequency prior
to the SCID clinical trials, resulted in several
studies on the mechanisms of viral vector

integration and oncogenic transformationin hematopoietic cells. Retroviral vector
integration into hematopoietic cells is semirandom, with the viral subtype, long terminal
repeats (LTRs), promoter, transgene andnumber of integrations per cell all potentiallyhaving an influence on either the site ofintegration or the genotoxic potential of
the gene therapy regimen. It has been clearly
demonstrated that gammaretroviral vectors
have a bias towards targeting enhancers andpromoter regions15, 16, whereas lentiviral
vectors show a preference for integrating
into active genes17, 18. Transcriptionally active
LTRs and strong viral enhancer/promoter
regions have been shown to substantially
increase the risk of oncogenesis19, 20, while
self-inactivating LTR vectors and wellinsulated or weaker promoters potentiallyconfer a reduced genotoxic risk. Despite
the prodigious amount of research that has
been performed on retroviral vectors21-23,
many of their mechanisms for cell targetingand integration are still poorly understood.
In the course of developing new therapeutic
lentiviral vectors for immune deficiencies such
as SCID-X124, recombination activating gene
1 and 2 (RAG)-SCID25, 26, lysosomal storage
disorder Pompe disease27 and mitochondrial
neurogastrointestinal
encephalomyopathy(MNGIE)28, we have tested a wide range of
expression cassettes differing with respect totheir selected promoters and transgenes, allincorporated in the same third generation
self-inactivating lentiviral vector backbone.
Hematopoietic stem and progenitor cells(HSC) were extracted from diseased mice,transduced and transplanted for long-term
follow up. Lentiviral vector integration sites

6

119Chapter 6(LVIS) were generated by linear amplification
mediated PCR (LAM-PCR)29 and highthroughput sequencing. LVIS were collectedfrom a variety of tissues, including lineage
negative (Lin-) bone marrow cells pretransplantation and bone marrow, spleen,
and thymus after transplant. Additional
lentiviral integrations were generated invitro by transduction of HSC derived frommouse bone marrow, rhesus bone marrow orhuman umbilical cord blood. In total, 12,236
unique LVIS were annotated from murine
experiments, including 4434 in vitro and 7802
in vivo integrations. Experiments in humanand rhesus HSC produced an additional 714
and 914 in vitro LVIS, respectively.

These integrations, generated using
the same transduction protocol in a single
laboratory, provided the opportunity toperform a comparative analysis of lentiviral
integration profiles across disease phenotypes,
hematopoietic cell types, and transgenes.
Here we have performed an in-depth analysisof oncogene prevalence, common integrationsite frequency, the tendency of LVIS to belocated near a TSS or near/within a Refseq
gene, Ingenuity Pathways Analysis and
HSC gene expression levels to determine
if any integration profile characteristics isover- or underrepresented in certain sample
sets, thereby generating further insight into
the integration profile of lentiviral vectors.

| |Results
Generation of an in silico dataset forcomparison
In silico datasets composed of 10,000,
3000, 1000, 500 or 300 randomly generated
integrations in the mouse genome weregenerated, using random chromosome andnucleotide position and weighted by the size ofthe chromosome. Using this method, randomchromosomal integration would produce
a consistent integration profile in datasets of
500 or more integrations (data not shown).
For comparison with the experimentallyproduced LVIS, a random dataset of 1000
integrations was chosen since similar in size
to most of the experimental datasets analyzed.

Integration site profiles of cultured
mouse, rhesus and human HPSC
Wild type Lin- mouse bone marrow (BM)cells, CD34+ cells derived from rhesus bonemarrow and CD34+ human umbilical cord
blood cells were cultured overnight with120

the pRRL.PPT.[SF or PGK].GFP.bPRE4*.
SIN lentiviral vector30, 31, at an MOI of 1-3 inorder to determine as to whether lentiviral
integration site preferences had a speciesspecific component. A total of 8 murine
samples, 10 rhesus samples and 4 humansamples were analyzed, yielding 1025, 914
and 714 unique integration sites, respectively.
The percentage of LVIS within 10kb of a gene
TSS (Table 1) ranged from 14.7% of mapped
LVIS for the human samples up to 20.3% for
the murine samples and the frequency ofintegrations within a gene or less than 10kb
from a 5’or 3’ gene terminus ranged from
69.6% (rhesus) to 73.4% (human). The numberof hot spots, termed common integration sites(CIS) scaled with the total number of LVIS
analyzed: a list of 3rd order or higher CIS foreach dataset can be found in Supplemental
Table 1. One 3rd order CIS oncogene (Mef2c)
was found in the mouse Lin- dataset.
The frequency of LVIS found near proto-

Hematopoietic stem cell lentiviral integration profiles
Table 1. LVIS recovered from cultured hematopoietic stem and progenitor cells.

Total LVIS
unique genes
LVIS within
10kb of TSSf
LVIS within
10kb of gene
intronic LVIS
uniqueoncogenes
total
oncogenes
percent
oncogenes
percent w/in
10kb of TSSf
percent w/in
10kb of gene

humana rhesusb mousec

Il2rgd

Rag1d

Rag2d

Gaad

In silico

714
642
105

914
802
142

1025
892
208

1043
893
176

148
141
25

402
373
78

188
181
36

1000
859
141

524

636

731

730

123

311

124

362

460
16

545
19

628
23

640
25

111
6

273
8

101
5

298
23

20

23

32

31

7

10

5

28

2.8%

2.5%

3.1%

3.0%

4.7%

2.5%

2.7%

2.8%

14.7%

15.5%

20.3%

16.9%

16.9%

19.4%

19.1% 14.1%

73.4%

69.6%

71.3%

70.0%

83.1%

77.4%

66.0% 36.2%

CD34+ cells derived from umbilical cord blood
CD34+ bone marrow cellscLin- bone marrow cells from wild type BALB/c micedLin- bone marrow cells from knockout micee1000 randomly-generated murine genome integrationsftranscription start site (TSS)a6boncogenes (as defined by the Retrovirus andtransposon Tagged Cancer Gene Database)in the rhesus, human and mouse datasets was
2.5%, 2.8% and 3.1%, respectively, which issimilar to the 2.8% frequency found in the insilico dataset. For each dataset, the Ingenuity
Pathways analysis was applied to the genes
within 10kb of an LVIS, and the top 20 results,
measured by p-value, listed in the Disease
and Functions analysis were compared. All
three species as well as the in silico dataset
had Cancer and Tissue Morphology among
the top 20 pathways (Supplemental Table 2).
The top functional pathway for the murine
Lin- samples was Cancer, with 46.4%
of mapped genes linked to this pathway(Supplemental Figure 1).

Lin- bone marrow cells derived frommurine models for SCID-X1 (Il2rg-/-), RAG1SCID (Rag1 -/-), RAG2 SCID (Rag2 -/-), andPompe disease (Gaa-/-), as well as wild type
BALB/c mice were cultured overnight withtherapeutic lentiviral vectors containing
the human version of the appropriate
transgene or GFP as a control. The cells
were subsequently cultured for 10-14 days
without viral vectors before DNA was
purified for LAM-PCR and LVIS analysis(Table 1). The frequency of oncogenes waslowest in the Rag2 samples (2.5%) andhighest in the Rag1 samples (4.7%), although
the difference in oncogene frequency
between these groups was not significant(p=0.176). The percentage of LVIS within
121

Chapter 6

10kb of a gene TSS or within 10kb of a geneappeared to be similar for all mouse diseasephenotypes analyzed.
The Il2rg-/- Lin- dataset had 1043 LVIS andthus the frequency of CIS could be directlycompared to the wild type mouse Lin- dataset(1025 LVIS). The frequency of integrations
flanking a TSS, gene, or oncogene were very
similar for the two datasets, with a slightly
higher frequency of LVIS within 10kb ofa gene in the wild type group. The Il2rg-/- datacontained no CIS oncogenes out of 54 CIS,
whereas the wild type group had one 3rd order
CIS oncogene (Mef2c) and a pair of second
order CIS oncogenes (Akap13 and Ghr) out
of 70 total CIS; however, this difference in
CIS oncogene prevalence was statistically notsignificant (p=0.261).

Comparison of integration profiles
between murine in vitro and in vivo
samples
Lin- bone marrow cells derived from
Il2rg-/-, Rag1-/-, Rag2 -/-, Gaa-/- and Tymp-/Upp-/- mice were cultured overnight withtherapeutic vectors or a GFP control
vector and subsequently transplanted into
the appropriate mouse disease phenotype.
Other wild type mice were transplantedwith wild type Lin- cells after overnighttransduction with a GFP control vector.
Transgene expression was driven by the spleen
focus forming virus (SF), ubiquitous promoter
elements phosphoglycerate kinase (PGK)
and ubiquitous chromatin opening element(UCOE), or cellular promoters including
the RAG1 promoter (RAG1p), the TCR Vβ6.7
gene promoter (TCRVβp), and the γ chain
promoter (γcPr). Genomic DNA was purified
from cultured Lin- cells and from bone122

marrow, spleen, and thymus tissue collected6-12 months after transplantation. Subsequent
LAM-PCR and sequencing of vector-genome
fusion sequences yielded over 2,000,000 raw
sequencing reads, which were annotated into
10,608 unique LVIS suitable for analysis.

Investigation of common integrationsites (CIS) in murine in vitro and invivo samples
Comparing the individual lists of CIS forthe various in vivo and in vitro datasets
revealed a number of CIS genes of 3rdorder or higher found in multiple datasets(Supplemental Table 3a), including protooncogene Mef2c. In total, 11 oncogenes were
found out of 264 total CIS of 3rd order orhigher (4.2% frequency of CIS oncogenes,
listed in Supplemental Table 3b).
Pooling all in vivo LVIS (consisting ofintegrations recovered from spleen, BM
and thymus, 7802 total LVIS) resulted in
the expected expansion of the number of CIS
found, with 522 CIS genes of 3rd order or higher
being returned. Similarly, the pooled murine
in vitro LVIS (consisting of 2806 total LVIS
recovered from cultured Lin- cells) contained
80 CIS of 3rd order or higher. 72% (58 out of80) of the Lin- CIS genes were also present as
CIS genes in the integrations derived frommurine cell types (Figure 1). Three of the top
five ranking CIS genes were the same for both
sample sets (Figure 2): a 139kb region on
chromosome 11 near Sfi1 (10th order in vivo
and 20th order in vitro), a 130kb region on
chromosome 5 near Uba6 (6th order and 21storder) and a 181kb region on chromosome 14
flanking Ngly1 (7th and 12th order). Anotherhigh-ranking CIS found in both datasets was
a 185kb region on chromosome 1, coveringHematopoietic stem cell lentiviral integration profiles

Figure 1. Common integration sites (CIS) of 3rd order or higher in murine in vitro vs invivo datasets. CIS of 3rd order or higher are reported in the overlapping ovals, with 58 shared CIS
genes. Word clouds below the ovals correspond to the relative frequency of each named gene in
the CIS pool (only the top 15most prevalent CIS genes in each dataset are shown).

most of gene Rgs18 (5th order and 10th order).
In general, these high ranking CIS were welldistributed between vectors, promoters andtransgenes and were not associated with any
specific murine phenotype (Figure 2).
Whereas no overall enrichment of
oncogenes was seen (frequency of LVIS
flanking oncogenes was 3.0% and 3.6% for
the in vitro and in vivo datasets, respectively),
the in vivo data had proportionally moreoncogenes present in high ranking CIS
compared to the in vitro data. The frequency
of oncogenes in the upper 25% of CIS(corresponding to 4th order or higher in vivo
and 3rd order or higher in vitro) was more
prominent in the in vivo dataset (5.9%,
compared to 3.8% for the in vitro dataset),
although also this difference was notstatistically significant (p= 0.589). Two

6oncogenes are present in these high ranking
CIS in both datasets: Lnpep and Mef2c.
The only tissue analyzed for all mouse
phenotypes was BM, so it was chosen as
the focus of further analysis (Table 2a).
The percentage of the LVIS in BM flanking
proto-oncogenes in the various diseasephenotypes ranged from 2.3% (Pompe
dataset) to 4.1% (wild type dataset), althoughthese differences were not significant. CIS of
3rd order or greater for these datasets can befound in Supplemental Table 4, and the listof CIS oncogenes of 3rd order or higher canbe found in Supplemental Table 5. Notably,
the percentage of LVIS within 10kb ofa gene was significantly lower for wild typemice using GFP vectors (59.3%) than in
any of the disease phenotypes (67.1% to71.1%, p<0.001 for all disease phenotypes

123

Chapter 6

Figure 2. Map of high ranking murine CIS found in both in vitro and in vivo samples.
Top: mouse chromosomal regions containing a high frequency of LV integrations in vivo and invitro are illustrated. Each marker (triangle) represents a single integration site. Bottom: distributionof LVIS among murine phenotypes for each of the highest-level CIS found in the in vitro andin vivo datasets.

compared to the wild type group). Separating
the disease phenotypes into GFP vector LVIS
and therapeutic vector LVIS (Table 2b) did
not significantly alter the LVIS profile, exceptthat the Pompe therapeutic vector subset hadsignificantly fewer LVIS within 10kb of a genethan the GFP vector subset (P = 0.04).
In order to determine as to whether in vivo
selection of clones in the BM during or after
engraftment skewed the integration profile,the LVIS of cultured Lin- and BM samples
for each of the four mutant mouse strains124

were compared (Table 2a). The percentages of
LVIS within 10kb of a gene TSS and within
10kb of a gene end were significantly lower in
LVIS recovered from wild type BM samples
than in LVIS recovered from wild type Linsamples (p <0.0001), but this difference was
not seen in the disease models. Significant
differences were not observed between
Lin- and BM samples in the frequency of
oncogenes (group averages of oncogene
frequency were 3.2% and 3.4%, respectively).
The integration profiles in spleen and thymus

Hematopoietic stem cell lentiviral integration profiles
Table 2a. LVIS recovered from Lin- and BM cells in murine disease models and WT

total LVIS
unique genes
percent oncogenes
percent w/in 10kb of TSS
percent w/in 10kb of gene
total LVIS
unique genes
percent oncogenes
percent w/in 10kb of TSS
percent w/in 10kb of gene

Il2rg

Rag1

Rag2

Gaa

TympUpp

WT

1043
893
3.0%
16.9%
70.0%
1372
1032
3.2%
14.1%
67.1%

148
141
4.7%
16.9%
83.1%
515
448
3.9%
21.4%
69.7%

402
373
2.5%
19.4%
77.4%
491
446
2.9%
17.7%
68.8%

188
181
2.7%
19.1%
66.0%
574
502
2.3%
19.9%
68.4%

ND
ND
ND
ND
ND
1075
885
3.9%
15.2%
71.1%

1025
892
3.1% Lin20.3%
71.3%
1402
981
4.1% BM
10.7%
59.3%

Table 2b. BM LVIS stratified by transgene
GFP transgene

total LVIS
unique genes
percent oncogenes
percent w/in 10kb of TSS
percent w/in 10kb of gene

Il2rg

Rag1

Rag2

Gaa

TympUpp

647
542
4.1%
14.0%
67.1%

31
26
0.0%
12.9%
74.2%

36
35
0.0%
13.9%
66.7%

364
321
2.2%
20.6%
71.4%

511
461
3.9%
15.3%
70.1%

6

Therapeutic transgene

total LVIS
unique genes
percent oncogenes
percent w/in 10kb of TSS
percent w/in 10kb of gene

Il2rg

Rag1

Rag2

Gaa

TympUpp

725
607
2.5%
14.0%
66.8%

484
423
4.1%
21.9%
69.4%

455
413
3.1%
18.0%
69.0%

210
196
2.4%
18.1%
62.9%

564
479
3.9%
15.1%
72.2%

Table 2c. Spleen and thymus LVIS stratified by transgene
GFP transgene

total LVIS
unique genes
percent oncogenes
percent w/in 10kb of TSS
percent w/in 10kb of gene

Therapeutic transgene

Il2rg

Rag1

Rag2

Il2rg

Rag1

Rag2

306
188
2.9%
20.9%
68.3%

81
60
0.0%
17.3%
64.2%

125
115
2.4%
13.6%
70.4%

52
31
3.8%
21.2%
78.8%

810
602
4.4%
21.7%
72.3%

1357
1080
4.5%
19.9%
73.0%

125

Chapter 6

were compared to BM in the same manner(Table 2c; the non-SCID phenotypes did nothave sufficient integrations for comparative
analysis). Significant differences between
these tissues were not found when comparing
the frequency of LVIS near genes, gene TSS
or oncogenes.

LVIS association to cellular gene
expression levels
Genes flanking LVIS were referenced
against an expression level binning databasegenerated via an Affymetrix mouse 430 2.0
gene array. Briefly, cDNA derived from mouse
Lin- Sca-1+cKit+ (LSK) cells after 2 days ofstimulation with growth factors was measured
on this whole genome array, and the resulting
gene expression values were sorted into fiveequally sized bins based on expression level.
Genes flanking an LVIS in the Lin- andBM datasets were referenced against thisdatabase and sorted into the appropriate bin.
The resulting binning chart (Supplemental
Figure 2) confirms that lentiviral vectors
prefer to integrate near or into active genes in
HSC. A similar expression pattern was seenin genes flanking in vitro LVIS (top) and invivo (bottom) integrations, suggesting that
LVIS near highly expressed genes undergo
minimal selective pressure. However, the top
10 overrepresented functions and diseases
based Ingenuity Pathways analysis thatappear for Lin- and BM datasets markedly
differed (Figure 3). The BM datasets showeda high frequency of Cancer, Hematopoiesis,
Cell to Cell Signaling and Interaction andCellular Development in their top 10 (these
diseases/functions were found in 4 out of 5
Lin- datasets). Conversely, the Lin- datasets
had a high frequency of Small Molecule

126

Biochemistry, Cell Cycle, and Cell Death andSurvival (each found in 4 out of 5 BM datasets).
Hematological System Development andFunction was frequently found in the top 10
of both groups: in all 5 BM datasets and in 4
out of 5 Lin- datasets.

Comparison of integration profiles:
GFP vs therapeutic transgenes
To determine as to whether insertion ofa therapeutic transgene results in a selective
advantage and LVIS bias after engraftment,
the integration profiles of BM samples from
diseased mice treated with therapeutic
vectors were compared with those of micetreated with GFP control vectors (Table 2b).
Significant difference in the frequency of
LVIS near genes, TSS or oncogenes were notseen between the GFP and the therapeuticgene datasets. Ingenuity Pathways Analysis
revealed that 6 out of the top 10 Ingenuity
Functions and Diseases, and 13 of the top
20, were shared between the GFP and IL2RG
groups (Figure 4).

Comparison of integration profiles:
SF vs eukaryotic promoter elements
The murine integrations analyzed here
include some studies using vectors containing
the SF promoter element in order to maximize
expression of the therapeutic transgene,as well as SF-GFP control vectors. The SF
promoter has been implicated in the onset of
oncogenic transformation in hematopoietic
cells19. The in vivo integrations collected frommurine disease models were stratified for SF vs
non-SF promoters (i.e., PGK, RAG1p, TCRβp,
UCOE and γcPr) to assess possible skewing
vector integration profiles in vivo. A total of3396 SF vector integrations from BM, spleenHematopoietic stem cell lentiviral integration profiles

6

Figure 3. Top 10 ranked IPA Diseases and Functions for murine Lin- and BM datasets. Thismatrix shows the top 10 highest significance (based on p-value) Ingenuity Diseases and Functions
for the LVIS genes in wild type (WT) and mouse disease models. The LVIS genes were stratified byintegrations recovered from in vitro (Lin- cells, left half of the figure) and in vivo (bone marrow, righthalf of figure) samples.

or thymus were compared to 3058 non-SF
vector LVIS (Table 3a). The frequency of LVIS
flanking oncogenes for the two datasets was
3.5% and 3.6% respectively and did not differ
significantly (p=0.904). Six of the top 10 IPA
diseases and functions were shared between
the SF and eukaryotic promoter integrations(Table 3b). Further stratifying the in vivo
LVIS pool by promoter (SF vs eukaryotic
promoters) and transgene (GFP vs therapeuticgene) resulted in no significant differences
between the 4 resulting data subsets(Table 3a). Stratifying only the BM
integrations by promoter returned the same
non-significant results (data not shown).

Analysis of LVIS found in leukemic
SCID mice
Experimental mice (primary and secondarytransplants) were monitored for adverse

127Chapter 6

Figure 4. Top Ingenuity Functional Pathways linked to in vivo samples containing GFP ortherapeutic transgenes. Matrix shows the top 10 highest significance (based on p-value) Ingenuity
Diseases and Functions for LVIS recovered from bone marrow in wild type (WT) and mouse diseasemodels. The BM LVIS genes were further stratified by integrations from GFP control vectors (leftpanel) or therapeutic vectors (right panel).

Table 3a. Integration profiles of in vivo LVIS stratified by promoter (SF viral promoter vs. eukaryotic
promoters) and transgene

total LVIS
percent oncogenes
percent w/in 10kb of TSS
percent w/in 10kb of gene

SF

euk.

3396
3.5%
19.0%
70.3%

3058
456
3.6%
1.8%
16.8% 19.7%
69.3% 70.0%

events. In the course of LV gene therapy
treatment of Il2rg-/- mice, 18 cases ofmalignant hematopoietic clonal expansion
were confirmed (Table 4 and Supplemental
Table 6 ), including 8 in mice treated with
128

SF-GFPeuk.-GFP SF-therap. euk.-therap.
1586
3.7%
15.6%
68.4%

2940
3.7%
18.8%
70.4%

1472
3.5%
17.9%
70.2%

IL2RG vectors (out of 117 primary mice,a 6.8% adverse event rate), 7 in mice treatedwith GFP vectors or vectors containing notransgene (out of 95 mice, 7.4%) and 3 in mice
given untransduced wild type Lin- cells (out

Hematopoietic stem cell lentiviral integration profiles
Table 3b. Top 10 Ingenuity Pathways Analysis functions and diseases for in vivo LVIS, stratified bypromoter (SF viral promoter vs. eukaryotic promoters)
SF promoter

eukaryotic promoters

Category

p-value

Category

p-value

Cellular Growth and Proliferation
Organismal Survival

5.06E-04
5.93E-04

1.21E-05
1.21E-05

Cell Death and Survival
Gene Expression
Hematological System
Development and Function
Hematopoiesis
Tissue Morphology

7.05E-04
8.50E-04
1.08E-03

1.25E-03
1.25E-03

Cell Cycle
DNA Replication, Recombination,
and Repair
CancerCellular Development
Hematological System
Development and Function
Hematopoiesis
Lymphoid Tissue Structure andDevelopment
Cellular Growth and Proliferation
Cell-mediated Immune Response

1.79E-03

Cellular Function and Maintenance 1.13E-04

Cell Cycle
DNA Replication, Recombination,
and Repair
Cancer

1.08E-03
1.08E-03

of 27 mice, 11.1%). 15 out of the 18 eventswere T-cell derived, including 13 CD4+CD8+
leukemia’s, and 2 single positives for CD4
and CD8. The remaining malignancies
consisted of one B220+CD19+, one CD11b+
and one Sca-1+ leukemia. All but one ofthese malignancies were donor-derived, as
determined by PCR through Y chromosome
chimerism levels. A clear correlation between
leukemiagenesis and promoter or transgene
was not observed; mice transplanted withuntransduced wild-type control cells had
the highest leukemic frequency. BM andspleen DNA from these mice was analyzed via
LAM-PCR to determine if certain integrations
were dominant, i.e. consisted of 50% or moreof the LVIS pool, which is often associatedwith dominant leukemic clones. Integrations
found in the leukemic mice were excluded
from the LVIS datasets to avoid skewing thatdata and increasing the prevalence of certain
oncogenes in the larger analysis.

1.82E-05
8.30E-05
8.30E-05
8.30E-05
8.30E-05
9.82E-05
1.13E-04

6

LAM-PCR analysis of these BM of thesemice revealed dominant LVIS flanking or in
the introns of oncogenes (Mef2c, Mll3, Ccnd2,
Pax5, and Aspscr1), transcriptional repressor
genes (Pum2 and Kctd1) and proposed tumor
suppressor genes (Pyhin1 and Shprh). It is
possible that some of these integrations may
have played a role in tumorigenesis in theseanimals, although no direct relation between
any IS and a leukemic event were confirmed.
Other mice with leukemia contained noprominent LVIS were found within 500kb
of any known proto-oncogene or tumorrelated genes.
Three leukemias were observed in control
Il2rg-/- mice transplanted with untransduced
wild type BALB/c Lin- cells, including the sole
recipient-derived malignancy with two inprimary transplants and one in a secondarytransplant. The primary leukemias were CD8+(donor derived) and CD4+CD8+ (recipient
129

130

2°
2°
2°
2°
2°
1°
1°
1°
1°
1°
1°
1°
1°
1°
1°
2°
1°
1°

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18

PGK.coIL2RG
PGK.GFPPGK.GFPPGK.GFPPGK.GFP
SF.coIl2RG
SF.Il2RG
SF.Il2RG
SF.empty
SF.empty
γcPR.coIL2RG
γcPR.coIL2RG
γcPR.coIL2RG
γcPR.coIL2RG
ycPR.GFP
none
none
none

LV vector
Il2rg-BALB/c
BALB/cBALB/cBALB/cIl2rg-Il2rg-Il2rg-BALB/c
Il2rg-Il2rg-Il2rg-Il2rg-Il2rg-BALB/c
BALB/cBALB/cBALB/cdonor cells* this mouse was given G-CSF mobilization in lieu of TBI
1° = primary transplant
2° = secondary transplant
TBI = total body irradiation
PB = peripheral blood
WBC = white blood cell
SF.empty = control vector, no transgene

transplant type

ID
6 Gy
6 Gy
6 Gy
6 Gy
6 Gy
0 Gy*
6 Gy
6 Gy
6 Gy
6 Gy
2 Gy
2 Gy
2 Gy
2 Gy
2 Gy
6 Gy
2 Gy
2 Gy

TBI dose
66
44
68
68
52
27
21
25
35
22
30
33
28
28
35
67
27
27

67.8
3.2
4.5
2.5
20.5
258
243
80
153
137
29.8
1.7
65.9
71
28.8
16.8
2.9
16.5

onset of illness PB WBC(weeks)count
CD4CD8
CD4CD8
CD4CD8
CD4CD8
CD4CD8
CD4CD8Sca-1
CD4CD8
CD11b
CD4CD8
CD4CD8
CD4CD8
CD4
CD4CD8
CD4CD8CD19CD8CD4CD8malignancy
phenotype
89
79
44
37
64
60.9
93
87
96
82
57
92
86
94
83
96
84
75

% phenotype
in BM

Table 4. Overview of leukemia phenotypes seen in Il2rg-/- mice during Lin- cell transplantation and gene therapy experiments

86%
87%
71%
71%
70%
59%
95%
62%
72%
75%
95%
57%
63%
82%
88%
95%
97%
12%

Y-chimerism(BM)

Chapter 6

Hematopoietic stem cell lentiviral integration profiles

derived), and the secondary leukemia had
a donor-derived, B220+CD19+ phenotype.
Four CD4+CD8+GFP+ leukemias were
found in mice treated with LV.PGK.GFP,
all in the secondary transplants of a single
experiment. This experiment involved analternate transplantation protocol: a very
low number of transduced wild type Lincells (3000 to 300 cells per mouse) wereexpanded in vitro for seven days, thenthe expanded cells were transplanted into 6
Gy conditioned Il2rg-/- recipients. Three ofthe leukemias shared an integration site on
chromosome 5 in intron 1-2 of gene Phtf2(putative homeodomain transcription factor2), suggesting that the pre-leukemic clone
originated in the primary cell pool prior totransplantation. No known oncogenes were
found within 500kb of these LVIS.

| |Discussion
Lentiviral vectors are a viable alternative togammaretroviral vectors for clinical genetherapy trials, offering a distinct integration
pattern which favors integrations in ornear active genes rather than near genetranscription start sites and have no propensity
to integrate near oncogenes. Previous
studies conducted with gammaretroviraland lentiviral vectors demonstrated thattranscriptionally active LTRs, such as thoseused in the initial SCID-X1 clinical trials,
are major determinants of genotoxicity19.
The same studies suggested that lentiviral
vectors require a 10-fold higher viral load to
trigger leukemogenesis than gammaretroviral
vectors, likely due to gammaretroviruses’
preference to integrate into the promoter
regions of active genes and into genes involvedin cell growth and cancer15.
Although many groups are investigating
lentiviral gene therapy vectors for treatment
of inherited diseases, assessing lentiviral
integration patterns is complicated bydifferences in vector design, transduction
protocols and integration site annotation
methodology, which induce variability andcomplicate direct comparison of lentiviral
integration profiles derived from different

sources. To enable large-scale comparisonof lentiviral integration profiles undera wide range of physiological settings, wepooled the total LVIS annotations from ourin vitro and in vivo experiments performedin the same lab. Nearly 14,000 lentiviral
integration sites have been annotated andanalyzed, including murine LVIS collected invitro and in vivo from immune deficiencies,
a lysosomal storage disorder, a mitochondrial
disorder and wild type mice, and LVIS fromtransduced human and rhesus CD34+ cells. By
stratifying the pooled LVIS based on various
parameters, we can observe which vector
features, such as promoter and transgene, may
act as selective forces on gene therapy-treated
HSC after transplantation and engraftment in
the bone marrow.
Comparison of LVIS generated in vitro inmouse, rhesus and human hematopoietic stemand progenitor cells confirms that the vector’s
integration preferences are not species
specific. The percentage of LVIS within 10kb
of a gene TSS ranged from 14.7% of mapped
LVIS for the human samples up to 20.3% for
the murine samples, a range consistent withprevious observations of LV vectors in human
HPSC and lower than the percentage reported

6131Chapter 6

for gammaretroviral vectors (29.3%)32.
The expected LV integration bias into generegions17 was observed, with the percentage
of LVIS within a gene or less than 10kb
distant ranging from 69.6% (rhesus) to
73.4% (human). Significant differenceswere not found between the three species
when comparing the frequency of LVIS neargenes, gene TSS, or oncogenes. The three
species also had a similar profile of Ingenuity
Functional Pathways, with all three having
an overrepresentation of genes involved incancer and tissue morphology.
It has been hypothesized that viral
integrations which improve the relative fitnessof transduced hematopoietic progenitor cells
in the competitive environment of the bonemarrow niche or thymus will give those cells
a competitive advantage. If this is the case,integration sites near proto-oncogenes orgenes involved in cell proliferation, would
more likely be found in vivo. The study
described here compared pre-transplant
Lin- samples to samples collected frombone marrow, spleen and thymus 6 months
or longer post-transplantation and revealed
similar frequencies in LVIS flanking an
oncogene. The expression profiles of genes
flanking LVIS in Lin- and BM samples weresimilar, with highly expressed genes being
favored. The relative frequency of LVIS within
10kb of a gene was lower in transplanted
cells than in Lin- cells for all phenotypes
except the Gaa-/- mice. The reduction in
LVIS near or in genes may result from someof the integrations disrupting gene function
and reducing the viability of the transduced
cells, an effect which would not be seen in
Lin- cells cultured for only a week. There isalso evidence that suggests post-transplantsite selection. Ingenuity Pathways analysis
132

of the genes flanking LVIS shows several
pathways more prevalent in BM samples
than in Lin- samples (Figure 3), including
Cancer, Hematopoiesis, and Lymphoid Tissue
Structure and Development. The nature ofthese overrepresented pathways suggeststhat selection for integrations flanking
cellular expansion genes could take placein vivo. It is also possible that there is no
selection in vivo, but that the differences
found are due to the differences in cyclingstate at transduction: the integration profilesof the Lin- cells analyzed here represent
integrations in cycling hematopoietic
progenitors as well as HSC, whereas the profile
observed in vivo is exclusively derived fromlong-term repopulating stem cells and theirprogeny, which are not cycling at the start ofovernight transduction.
The frequency of common integrationsites (CIS) varied based on the size ofthe dataset analyzed, but in all cases was 3-to
4-fold higher than the expected frequency of
CIS in datasets of comparable size based onmathematical models of random integrationor on in silico data 33. This further emphasizes
that lentiviral integration patterns are only
semi-random and that LV integration hot
spots are prevalent in murine HSC. A majorityof the CIS genes identified in the Lin- samples
were also CIS genes in the post-transplant
samples, indicating that these genes are locatedin areas of frequent lentiviral integrations.
Two hot spots in particular, located in ~140kb
spans in genes Sfi1 and Uba6, were foundfrequently across multiple cell types, vectors
and disease phenotypes (Figure 2). Sfi1, oneof the most commonly found LVIS genes inboth the in vitro and in vivo data, is involved
in hematopoietic differentiation and has been
noted previously as a lentiviral integration

Hematopoietic stem cell lentiviral integration profiles

hot spot in both murine Lin- BM cells34, 35 andhepatocytes36. Uba6 is an ubiquitin-activating
gene, and LV integrations near this gene havebeen reported before in a tumor-prone mousemodel34. To the best of our knowledge, LV
integration hot spots found on chromosomes
1 and 14 have not been previously associatedwith viral integration.
Two CIS oncogenes were found in boththe in vitro and in vivo murine integrations:
Mef2c on chromosome 13, and Lnpep onchromosome 17. Both of these oncogenes
were high-order CIS in vivo (12th order and9th order, respectively). An integration near
Mef2c was also found in a Il2rg-/- mousetransduced with a LV.SF.IL2RG vector which
had an unusual Sca-1+ leukemic phenotype.
Mef2c is a CIS for gammaretroviral vectors37,
38, which suggests a role in hematopoietic
development. Additionally, this gene has beenpreviously identified as a possible contributor
to T-ALL in humans38.
The Lin- data had one additional
CIS oncogene, Akap13, which was not
a CIS in the transplanted tissues (only 2
integrations were found flanking this genein vivo). There were 47 CIS oncogenes inthe transplanted cells that were not present
as CIS in the Lin- cells, including an 8th order
CIS in the Mds1 and Evi1complex locus(MECOM), an integration site commonly
found in transduced mouse hematopoietic
progenitors39. Overall, the frequency of CIS
oncogenes was somewhat lower for the Linthan the transplanted LVIS (3.3% vs 3.9%),
although not significantly (p=0.738).
Comparing the LVIS profiles of therapeutic
vectors to GFP control vectors revealed
no evidence of integration site skewing
in the frequency of integrations flanking
gene TSS or oncogenes and did not affectthe overrepresented Ingenuity Functional
Pathways (Figure 4). Stratifying the LVIS
profiles of mouse disease models by promoter
and comparing vectors where transgene
expression is driven by the SF viral promoter
versus vectors using a eukaryotic promoter
also showed no significant differences in
the function of flanking genes or the frequencyof nearby oncogenes. These findings indicatethat the promoter and transgene incorporated
by lentiviral vectors has little or no effect on
LV integration profiles.
Following the initial reports of leukemiasin SCID-X1 gene therapy trials11, 12, the MFG
Moloney-gammaretrovirus derived vector
used in these trials was implicated as having
a role in oncogenesis. The case against
viral enhancer/promoters was made due tothe strength of activation of nearby genes
and its possible influence on the integrationsite profile 18, 20. In vitro immortalisation
assays seemed to confirm the higher
genotoxic risks of viral promoters, suchas the SF promoter, compared to weakereukaryotic promoters 20, 40.
In our experiments with SIN lentiviral
vector gene therapy for murine SCID-X1,
the choice of promoter seemed to have no effecton the rate of leukemogenesis. Mice treatedwith the SF promoter had a leukemia rate of
6%, similar to the rates seen in eukaryotic
promoters PGK (9%) and γcPr (6%), while
the highest leukemia rate (11 %) was observedin recipients of untransduced wild-type cells.
While it is feasible that the additional risks
associated with the SF vector in vitro are
reduced in vivo due to regulation of the bonemarrow niche, it is also possible that ourmouse model assay, with its limited number ofmice per group, is not sufficiently sensitive todetect differences in the oncogenic potential

6133

Chapter 6of the various promoters. However, the more
likely explanation involves leukemogenesis dueto the Il2rg-/- phenotype independent of vector
integrations. Il2rg-/- mice are known to beprone to leukemia development41, particularly
after HSC transplantation. The large majority(15 out of 18) of the observed leukemia’s
occurred in the T cell lineages. This was
highly significant and suggests that the T celllineages are especially prone to oncogenesis
in this setting. It is noteworthy that all 5
leukemias seen in the human SCID-X1 genetherapy trials were T-ALL, as were 6 out of
7 leukaemias seen in the trial for WiskottAldrich syndrome (WAS)14: both WAS
and SCID-X1 are T- immunodeficiencies.
Similarly, the two adverse events seen in

the chronic granulomatous disease (CGD)
clinical trial were myelodysplasias13. It
is possible that corrected hematopoietic
progenitor cells belonging to the deficient cell
type are subjected to additional proliferative
stress and therefore more susceptible totransformative events, particularly when
this stress is combined with disruptive viral
integrations near key oncogenes.
Overall, these data suggest that lentiviral
integration profiles are stable across species
and in mice with a wide range of phenotypes,
and that LV vector HSC gene therapy has a low
risk of integrating vectors near oncogenes
similar to that found in in silico and random
integration datasets.

| |Materials and Methods
Animals
Wild-type BALB/c or C57BL/6, and C57BL6-Rag1-/-26, BALB/c-Rag2-/-25, BALB/c-Il2rg-/-24,
FVB/N-Gaa-/-42 and C57BL/6-Tymp-/-/Upp-/(MNGIE) mice were used for gene therapy
studies as previously described. Purpose
bred rhesus monkeys (Macaca mulatta)
aged 2 to 3 years were housed in stainlesssteel cages, preferably in small peer groups
and provided with normal daylight rhythm,
conditioned to 20°C with a relative humidityof 70%. All mice and rhesus monkeys werebred in the Experimental Animal Centerof Erasmus MC. Housing, experiments
and all other conditions were approved by
the institutional Animal Ethical Committeeof Erasmus MC in accordance with legislationin the Netherlands.

Lentiviral vectors
The third generation self-inactivating (SIN)
HIV-1 lentiviral vector (LV) pRRL.PPT.
134

SF.GFP.bPRE4*.SIN30, 31 backbone was usedand derivatives were constructed using aspreviously described24-27.
Therapeutic

promotercassettes

weredivided in virus promoter (spleen focus
forming virus; SF), ubiquitous promoters(UP) and cellular restricted promoters (CP).
UP included the house keeping promoter
phosphoglycerate

kinase(PGK),

anda ubiquitous chromatin opening element(UCOE)43. The CP used were the human
RAG1 promoter (RAG1p), the TCR Vβ6.7
gene promoter (TCRVβp), and the γ chain
promoter (γcPr)26, 44. The CP promoterswere made by PCR amplification of humangenomic DNA.
The SF.GFP construct was used for invitro transduction of murine, rhesus andhuman HSC and in vivo studies in wild type
BALB/c mice. The full list of lentiviral vector
constructs used for in vivo murine experiments
and the disease backgrounds can be found inHematopoietic stem cell lentiviral integration profiles

Supplemental Table 7. Several therapeutic
genes were subjected to codon optimizationof transgene cDNA (nomenclature “co”)
to improve transcription and translation45.
Codon optimization was performed by
GeneOptimizer® software (GeneArt AG,
Regensburg, Germany) or OptimumGene™ Codon Optimization (Genscript, USA).

Transduction of human, rhesus andmouse hematopoietic progenitors
Lin- cells from male mice were purified bylineage depletion (Lin-) (BD Biosciences,
Santa Clara, CA). Rhesus CD34+ HSC
were obtained from freshly collected or
cryopreserved humeral bone marrow aspirants
followed by positive selection for the CD34
antigen. Human CD34+ HSC were obtained
from freshly collected or cryopreserved
umbilical cord blood followed by positive
selection for the CD34 antigen. All cells were
cultured in serum free modified Dulbecco’s
medium with supplements as described 46
and growth factors murine stem cell factor(mSCF) 100 ng/mL, human FMS-liketyrosine kinase 3-ligand (hFlt3-L) 50ng/ml
and murine thrombopoietin (mTPO) 10ng/
ml for murine cells. Human SCF was used
for human and rhesus cells, and human TPO
and rhesus TPO was used for species specificcells at similar concentrations as for murine
cells. All hematopoietic cells were transduced
overnight at various cell densities. Vector
dose varied based on the experiment, witha range 0.2 to 5.9 vector copies per Lin- cell, as
measured by qPCR. Subsequently, cells were
cultured for 5-9 days before harvesting Linsamples, ormurine cells cultured overnightand subsequently injected in a range of 1×106
to 1×104 Lin- cells into the tail vein of 6 Gyor 2 Gy irradiated female recipients. Mice

were followed for six to twelve months andsubsequently DNA was purified from bonemarrow, spleen and thymus.

LAM-PCR and integration siteanalysis workflow
High-resolutionbylinearinsertion-site

analysis

amplification–mediated

PCR(LAM-PCR) of vector-genome border regions
was performed as previously described29. Restriction enzymes Tsp509I or MseI

were used with the lentiviral (HIV) primer
set. LAM-PCR products were pooled andsequenced at GATC Biotech (Konstanz,
Germany)

andsubsequently

annotated.

Briefly, viral sequences were trimmed andthe remaining genomic sequences aligned
to the appropriate genome via BLAST using

6

previously described parameters for to
distinguish bona-fide integration sites7, withthe following exceptions: MAVRIC47 was usedas the annotation software with the following
parameters: no repeat masking, minimum
sequence length = 20 bp, minimum BLAST
e-value = 0.01, max distance from integrationsite to look for genes = 100kb. Sequenceswere aligned to the murine genome via
BLAST (v.37) and nearby genes identified
using Ensembl version 59. Sequences witha sequence count <2 were excluded fromfurther analysis. Each LVIS was annotatedwith a single, nearest Refseq gene, which
was subsequently used for downstream
analysis. Genes nearest to an integration sitewere flagged as proto-oncogenes based ona list of 528 mouse common integration site
genes from the Retrovirus and transposon
Tagged Cancer Gene Database. Common
integration sites (CIS) were determined basedon established methods48.
135Chapter 6

Statistics
P values for comparisons of the frequency of
oncogenes, LVIS near TSS, and LVIS within
10kb of a gene were calculated using the Chisquare test or Fisher’s Exact test (2-sided),with Bonferroni’s correction used to adjust
the threshold p value (α) where applicable.

P-valuesforoverrepresented

pathways

calculated

functional

automaticallyusing

Ingenuity’s software (Fisher’s Exact test) andadjusted for multiple comparison analysis
using Bonferroni’s correction (calculatedon the basis of 75 functional groups foreach analysis).

| |Acknowledgements
The authors acknowledge Dr. R. Martí and
Dr. J. Barquinero (Mitochondrial Disorders
Unit and Gene and Cell Therapy Unit, Valld’Hebron Institut de Recerca, Universitat
Autonoma de Barcelona, Barcelona, Spain)
for providing the Tymp-/-Upp-/- mice, Dr. N.
Berinstein (Department of Immunology,
University of Toronto) for the RAG2 promoter
plasmid, and Prof. F.J.T. Staal and Dr. K. PikeOverzet, Dept. Stem Cell Biology, Leiden
University Medical Center, for assistancein the microarray analyses. Funding wasprovided by the European Commission’s 5th,
6th and 7th Framework Programs, Contracts

136

Q L K 3 - C T- 2 0 0 1 - 0 0 4 2 7 - I N H E R I N E T,
L S H B - C T - 2 0 0 4 - 0 0 5 2 4 2 - C O N S E R T,
LSHB-CT-2006-19038-Magselectofection,
Grant Agreement 222878-PERSIST and
Grant agreement 261387 CELL-PID, by
The Netherlands Organisation for Scientific
Research (NWO), Grant 021.002.129,
and by the Netherlands Health Researchand Development Organization ZonMW(Translational Gene Therapy program projects
43100016 and 43400010). The authors
also gratefully acknowledge stipends fromthe Join4Energy Foundation (MWH) and
the Ride4Kids Foundation (RY).

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van Til, NP, de Boer, H, Mashamba, N,
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Cesana, D, Ranzani, M, Volpin, M,
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35.

Maetzig, T, Brugman, MH, Bartels, S, Heinz,
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36.

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Bosticardo, M, Ghosh, A, Du, Y, Jenkins,
NA, Copeland, NG, and Candotti, F (2009).
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reproducible and stable transgene expression
in hematopoietic cells. Blood 110: 1448-1457.

gene transfer induces oncogene activationand immortalization of primary murine bonemarrow cells. Mol Ther 17: 1910-1918.
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Dave, UP, Akagi, K, Tripathi, R, Cleveland,
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Markiewicz, S, Bosselut, R, Le Deist, F, deVillartay, JP, Hivroz, C, Ghysdael, J, et al.
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39.

Modlich, U, Navarro, S, Zychlinski, D,
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Zychlinski, D, Schambach, A, Modlich, U,
Maetzig, T, Meyer, J, Grassman, E, et al.
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the genotoxic risk of integrating gene vectors.
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Shou, Y, Ma, Z, Lu, T, and Sorrentino, BP (2006).
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Neelis, KJ, Hartong, SC, Egeland, T, Thomas,
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47.

Huston, MW, Brugman, MH, Horsman, S,
Stubbs, A, van der Spek, P, and Wagemaker,
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48.

Deichmann, A, Brugman, MH, Bartholomae,
CC, Schwarzwaelder, K, Verstegen, MM, Howe,
SJ, et al. (2011). Insertion sites in engrafted
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gene therapy. Mol Ther 19: 2031-2039.

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43.

Bijvoet, AG, van de Kamp, EH, Kroos,MA, Ding, JH, Yang, BZ, Visser, P, et al.
(1998). Generalized glycogen storage andcardiomegaly in a knockout mouse model of
Pompe disease. Hum Mol Genet 7: 53-62.
Zhang, F, Thornhill, SI, Howe, SJ,
Ulaganathan, M, Schambach, A, Sinclair, J,
et al. (2007). Lentiviral vectors containing anenhancer-less ubiquitously acting chromatin

6139

Chapter 6| |Supplementary information

Supplementary figure 1. Ingenuity Functional Pathways analysis of HSC integration sites.
Overrepresented Ingenuity functional pathways for genes flanking integration sites in hematopoietic
stem and progenitor cells (HPSC) purified from murine, rhesus and human sources transduced with
GFP lentiviral vectors. Significance is shown on y-axis as –log10 P value. The base significancethreshold of P < 0.05 was adjusted for multiple comparisons using the Bonferroni correction to reacha new threshold of P < 0.00022 (75 functional classes and 3 cell types = 225 function comparisons).

140

Hematopoietic stem cell lentiviral integration profiles

6

Supplementary figure 2. Gene expression binning data. Gene expression values of Lin- Sca-1+
c-Kit+ cells after two days of stimulation, as measured on an Affymetix mouse 430 2.0 array, were
sorted into 5 equally-sized bins based on their relative expression levels. The genes nearest to an
integration site in one of the lineage negative (Lin-) or bone marrow (BM) datasets are used togenerate a histogram based on this binning profile. Highly expressed genes are much more likely tocontain nearby integrations in both the Lin- and BM samples.

141

Chapter 6
Supplementary table 1. CIS of 3rd order or higher in cultured hematopoietic stem/progenitor cells

species

CIS
Chromorder osome Loci

Genes

mouse
mouse
mouse
mouse
mouse
mouse
mouse
mouse
mouse
mouse
rhesusrhesusrhesusrhesusrhesus
human

4
3
3
3
3
3
3
3
3
3
4
3
3
3
3
3

2700029M09Rik;Clcn3
D1Ertd622e;Gin1
Akt3
Uba6
Ica1
Smarcad1;Hpgds
Plscr1;1700057G04Rik
Reep3
Mef2c*
Gm4117
LOC719197
RAPGEF6PCM1LRRCC1MBD2ANTXR2

8
1
1
5
6
6
9
10
13
13
2
6
8
8
18
4

63392605;63405611;63437922;63453218
99612093;99640538;99649662
179046612;179054779;179082714
86575222;86577004;86583707
8681945;8699661;8707816
65022182;65032843;65058002
92153249;92154125;92190992
66642587;66650208;66660333
83673980;83674833;83688671
89998176;90012865;90043437
1954335;1959596;1993923;2030622
128127211;128129891;128133390
17822238;17829654;17859303
87646981;87665223;87665361
47431494;47461444;47461626
80874986;80876679;80877724*RTCGD listed proto-oncogene.

Supplementary table 2. Ingenuity pathways analysis of overrepresented diseases and functions in
cultured HSC transduced with lentiviral vectors
Top 20 disease or biological function

found inCancerTissue Morphology
Gene Expression
Cardiovascular System Development & Function
Organismal Development
Cellular Development
Cell Death & Survival
Cellular Movement
Cellular Assembly & Organization
Cell Morphologyall
all
H, R, S
H, M, S
H, M, S
H, M, S
H, R, S
M, R, S
M, R, S
M, R, S

H=human, M=murine, R=rhesus, S=in silico datasets

142

Hematopoietic stem cell lentiviral integration profiles
Supplementary table 3a. CIS found in multiple murine datasets (3rd order or higher)
CIS gene

Chromosome

CIS order

Range of loci

Dataset

Uba6

5

Gtdc1

2

Mef2c

13

Plxdc2

2

Mrps9

1

Ube3a

7

Pcdh9

14

Gin1

1

Samsn1

16

Sfi1

11

Cntnap2

6

Slc4a7

14

6
3
3
3
4
3
3
5
3
4
3
3
3
3
3
3
3
3
3
3
3
10
4
3
3
5
3

86569855-86582241
86567296-86590346
86575222-86583707
86562050-86583025
44703455-44758157
44742547-44776921
44550627-44550702
83640034-83757178
83673980-83688671
16422537-16484631
16402704-16428376
42912652-42929105
42923559-42931481
66528779-66562531
66524455-66547103
94187748-94187763
94196123-94197963
99612093-99649662
99661378-99661493
75892377-75911575
75945565-75945918
3031603-3110289
3039287-3110058
45043093-45065563
45228340-45256599
15548060-15598104
15470107-15470296

Il2rg in vivo
Rag2 in vivo
WT in vitro
WT in vivo
Rag2 in vivo
UppTP in vivo
Rag1 in vivo
Il2rg in vivo
WT in vitro
Il2rg in vivo
Rag2 in vivo
Il2rg in vivo
Il2rg in vitro
Il2rg in vivo
Rag2 in vivo
Il2rg in vivo
Rag1 in vivo
WT in vitro
Rag1 in vivo
Rag2 in vivo
Rag1 in vivo
Rag2 in vivo
Il2rg in vitro
Rag2 in vivo
WT in vivo
WT in vivo
Rag2 in vivo

6143

Chapter 6
Supplementary table 3b. CIS oncogenes found in mouse datasets (3rd order or higher)

144

CIS oncogene

CIS
order

Chromosome

LocidatasetArid1aCdk6
Coro1aGimap7
Ikzf1
Lnpep
Lrmp
Mef2c
Plag1
Sdccag8
Zfp608

4
3
3
4
3
3
3
5;3
8
4
3

4
5
7
6
11
17
6
11
4
1
18

133273225-133273420
3375849-3381855
133862044-133863162
48659062-48679534
11604680-11624778
17750933-17764326
145076099-145076149
83640034-83757178
3858379-3862732
178831743-178832157
55122201-55123377

Rag1 in vivo
Rag1 in vivo
Rag1 in vivo
Rag1 in vivo
Rag1 in vivo
WT in vivo
Rag2 in vivo
Il2rg in vivo; WT in vitro
WT in vivo
Il2rg in vivo
Rag2 in vivo

Hematopoietic stem cell lentiviral integration profiles
Supplementary Table 4. all CIS of 3rd order or higher in murine phenotype BM samples
phenotypeCIS order

chromosome genes

loci

Il2rg

6
5
5
4
4
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
4
3
3
3
3
3
3
3
3
3
3
3
3
3
3
8
6
5
4
4
4
4

5
5
5
3
12
1
1
1
4
7
11
11
12
13
14
17
18
18
19
19
X
13
1
2
8
9
10
11
12
X
4
14
14
17
17
17
4
7
14
1
8
13
17

86569855-86582241
16095587-16294275
90216928-90284089
30117285-30119266
52693480-52762846
42912652-42929105
99630458-99661078
99908290-99908664
52459854-52487919
66528779-66562531
3031770-3078236
69550965-69551453
37623133-37669935
83640034-83681077
79940798-79941001
33891546-33892143
75574839-75574919
89049399-89050188
37025311-37035490
39020441-39034829
140762892-140788621
13744606-13790854
89281135-89281173
44742547-44776921
42357497-42390158
3428516-3477478
50320516-50359114
49496481-49503060
78429053-78467732
126677649-126719423
133273234-133273420
37721390-37729164
119414191-119414225
21825425-21869361
35368970-35385457
64672665-64673226
3858379-3862732
34930305-35091597
15548060-15598104
84739858-84766670
55188886-55262672
34082481-34155957
89494826-89573458

TympUpp

Rag1

wild typeUba6
Hgf
Adamts3
Mecom
Coch; Strn3
Mrps9
Hisppd1
Pam
Smc2
Ube3a
Sfi1
Polr2a4930579E17Rik
Mef2c
Elf1
Gm17251
Smad7
Socs6
Btaf1
Hells
Alg13; Trpc5
Lyst; Gng4
Kcnj13
Gtdc1
Pcm1
Cwf19l2; Gucy1a2
Ascc3
Scgb3a1
Fut8
Diap2
Arid1aGcap14
Dnajc3
Zfp760Nfkbil1; Bat1a
Pja2
Plag1
Uba2
Slc4a7
Trip12
Vegfc
Nqo2; Ripk1; Bphl
Fshr

6

145

Chapter 6
Supplementary Table 4. (continued)
phenotypeCIS order

chromosome genes

loci

4
3
3
3
3
3
3
3
3

18
1
2
5
5
6
17
17
18

46919714-46942581
145537654-145545756
105761317-105761521
43735180-43759290
86562050-86583025
45228340-45256599
17750933-17764326
26872812-26876216
90689757-90706582

Ap3s1
Cdc73
Immp1lCpeb2
Uba6
Cntnap2
Lnpep
A930001N09Rik
Tmx3

Supplementary table 5. CIS oncogenes found in murine in vitro and in vivo LVIS datasets

146

phenotypeoncogene

CIS order chromosomelociIl2rg in vivo
Il2rg in vivo
murine in vitro
Rag1 in vivo
Rag1 in vivo
Rag1 in vivo
Rag1 in vivo
Rag1 in vivo
Rag2 in vivo
Rag2 in vivo
WT in vivo
WT in vivo

Mef2c
Sdccag8
Mef2c
Arid1aGimap7
Cdk6
Coro1aIkzf1
Lrmp
Zfp608
Plag1
Lnpep

5
4
3
4
4
3
3
3
3
3
8
3

83640034-83757178
178831743-178832157
83673980-83688671
133273225-133273420
48659062-48679534
3375849-3381855
133862044-133863162
11604680-11624778
145076099-145076149
55122201-55123377
3858379-3862732
17750933-17764326

13
1
13
4
6
5
7
11
6
18
4
17

Hematopoietic stem cell lentiviral integration profiles
Supplementary table 6. details of LVIS found in Il2rg-/- leukemias

ID # vector

integrations % clonal chromper BM cell dominance osome LVIS locus flanking gene

1

1.1

PGK.
coIL2RG

99

112

PGK.GFP

2.4

92

5

3

PGK.GFP

3.8

37

5

31

11

24

5

120421360 Arl16
Gm17586
Npb
Anapc11
Gm11789
Fam195bPpp1r27
P4hb
Arhgdia
Gcgr
Alyref
Pcyt2
Sirt7
Slc25a10
Mafg
Mrpl12
Pycr1
Myadml2
Hgs
20378460 Tmem60
Phtf2
20378460 Tmem60
Phtf269550965 Mpdu1
Cd68
Eif4a1
Senp3
Tnfsf13
BC096441
Zbtb4
Polr2aAmac1
2010012P19Rik
Chrnb1
Fgf11
Tmem102
Mir1934
4933402P03Rik
Sox15
Spem1
90217670 Adamts3

distancefrom IS togene 5’ end
92.6 kb US
73.7 kb US
48.4 kb US
38.4 kb US
28.4 kb US
10.5 kb US
9.0 kb US
13.1 kb DS
21.4 kb DS
29.3 kb DS
38.2 kb DS
57.8 kb DS
65.1 kb DS
68.2 kb DS
73.4 kb DS
75.4 kb DS
83.6 kb DS
88.0 kb DS
92.4 kb DS
9.6 kb US
9.4 kb DS
9.5 kb US
9.4 kb DS
74.9 kb US
71.4 kb US
65.1 kb US
55.5 kb US
51.8 kb US
41.4 kb US
28.4 kb US
21.1 kb DS
24.4 kb DS
55.3 kb DS
58.4 kb DS
64.3 kb DS
68.1 kb DS
74.4 kb DS
80.9 kb DS
82.1 kb DS
84.6 kb DS
94.7kb DS

6147

Chapter 6
Supplementary table 6. (continued)

ID # vector

integrations % clonal chromper BM cell dominance osome LVIS locus flanking gene

4

PGK.GFP

2.6

5

PGK.GFP

3.1

80
19
92

19
4
5

12 γcPR.
coIL2RG

1.6

68

2

13 γcPR.
coIL2RG
14 γcPR.
coIL2RG

1.8

22
99

11
18

27937685 Rfx3
111458245 Spata6
20378460 Tmem60
Phtf2
8728063
Sdc1
Pum2
83644300 Mef2c*75428013 Pdcd10
Serpini124812154 Galntl5
Galnt11Mll3*108962025 Mettl1512297464 Utrn
8632961
Pmm2
Tmem186
Carhsp1
29899713 Mecom
n/an/a154308268 Klhl34
107028782 Papola
Ak7
127152223 9630033F20Rik
Ccnd2*
9330179D12Rik
32444912 Mir1954
Eng
Ak1
St6galnac6St6galnac4
Pip5kl1
Dpm2
Fam102a
Fpgs
43946382 Il12b
15379080 Kctd1

6SF.coIL2RG 1.1

97

12

7

SF.IL2RG

2.2

72
10

13
3

8

SF.IL2RG

2.8

52

5

SF.poly

2.9

22
18
69

2
10
16

10 SF.poly
11 γcPR.
coIL2RG

0
1.4

30
n/a
61
13

3
n/a
X
12

10

6

1.7

58

4

44798766

15 γcPR.GFP

0.8

33
99

10
16

957659768844799* known proto oncogene; # tumor suppressor gene

148

Pax5*
Gm12463
Zcchc7
Stxbp5
Rbfox1

distancefrom IS togene 5’ end
10.5 kb US
65.7 kb DS
9.5 kb US
9.4 kb DS
50.0kb US
47.1kb DS
1.1 kb DS
67.4 kb US
66.5 kb DS
124.8 kb DS
83.4 kb DS
192.3 kb DS
158.9 kb DS
291.6 kb DS
4.7 kb US
4.6 kb DS
39.1 kb DS
547kb DSn/a51.9 kb DS
10.3 kb DS
84.6 kb DS
92.7 kb US
51.2 kb US
52.8 kb DS
63.2 kb US
57.5 kb US
32.7 kb US
10.6 kb US
2 kb DS14.3 kb DS
18.2 kb DS
53.8 kb DS
114.7 kb DS
267.2 kb DS
69 kb US74.5 kb US
32.8 kb US
30 kb DS75 kb DSHematopoietic stem cell lentiviral integration profiles
Supplemental table 7. list of LV vectors used in each murine phenotype
Phenotype

Vector

Il2rg

PGK-GFPPGK-IL2RGco
SF-IL2RG
SF-IL2RGcoγcPr-GFP
γcPr-IL2RGco
PGK-GFPPGK-TYMPPGK-TYMPco
SF-TYMPco
PGK-GAAco
SF-GAAco
SF-GFP
RAG1p-RAG1co
SF-GFP
SF-RAG1co
TCRβp-RAG1coγcPr-RAG1co
SF-RAG2co
UCOE-GFPUCOE-RAG2co
PGK-GFPSF-GFP

TympUpp

Gaa

Rag1

Rag2

BALB/c6

149

General discussion

7

General discussion| |Lentiviral vector gene transfer into
hematopoietic stem cells
Hematopoietic stem cell gene therapy(HSCGT) has been successfully used inclinical trials for a wide range of inherited
hematological, metabolic, and immune
deficiency disorders.1 Recently, the first ex
vivo HSCGT drug (Strimvelis) was approved
for marketing in Europe for the treatmentof adenosine deaminase severe combined
immunodeficiency (ADA-SCID). The productis made of autologous hematopoietic
stem cells (HSCs) transduced witha gammaretroviral (γ-RV) vector containing
ADA, therefore an excellent personalized
treatment of ADA-SCID patients for whoma matched donor is lacking. Due to the highly
proliferative capacity of hematopoietic stemand progenitor cells (HSPCs), RVs have been
employed in HSC gene transfer protocols
for their capacity to integrate into the host’s
genomic DNA and propagate transgene
expression into progenitors and mature cells.
Pre-clinical studies resulted in sufficient proof
of concept 2,3 to move towards clinical trials
for primary immunodeficiencies using γ-RV
vectors. These trials showed the efficacy ofthis gene therapy approach demonstrating
phenotypiccorrection
andclinical
improvements following the restoration of
the cellular, and in some, also the humoral
immunity.4,5 However, a large proportion of
the X-linked SCID and Wiskott Aldrich γ-RV
trials reported hematological malignancies
related to the upregulation of protooncogenes close to the vector insertion
sites.6-11 Therefore, efforts were directed
towards the development of third
generation self-inactivating (SIN) lentiviral(LV) vectors as an alternative vector system forreducing the risk of insertional oncogenesis.12
Another advantage of using LV vectors isthat the proliferation of HSPCs during genetransfer is not a requirement, in contrast to
γ-RV vectors.13 The current protocols using
RV (both γ-RV and LV) employ two roundsof transduction, high vector doses (MOI100), and relatively high concentrations ofmultiple cytokines.14,15 These conditions,however, could affect the efficacy and safety
outcomes of HSCGT, by increasing the risk ofinsertional mutagenesis of the genes involvedin cell-cycling and by compromising
the long-term repopulation capacity of genemodified HSCs. Our findings show efficientgene transfer into HSPCs during a shorttransduction time by increasing the target
cell density with a proportional increase of
LV particles. Addition of thrombopoietin(TPO) alone was sufficient in maintaining
the in vivo repopulation capacity and ensuring
efficient transduction at the same time. Ourevaluation of human HSPCs was limited toin vitro assessments, and further experiments
might be required to estimate the efficacy of
prolonged gene marking and repopulation
capacity through transplantation into
the humanized mouse model.16 Additionally,of interest is the application of this protocol
in mouse models of human diseases
to evaluate the contribution towards
phenotypic correction; performance ofthe transductions in microfluid-basedtransduction system, wherein the probabilityof infection is increased;17 and combination
with prostaglandin E2 stimulation leading

7

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Chapter 7

to effective LV transduction of HSCs during
short culture time (less than 38hrs).
Theseadvancements

at18

generatinga clinically viable and feasible protocol for LV
transduction of HSPCs could be combinedwith strategies to enhance the expansion ofgene modified HSPCs. Ex vivo expansion
would support the maintenance of the (stemness) of the gene modified HSPCs, enable
selection of HSPCs with integrated LV
vector copies and transgene expression prior
to transplantation, and the evaluation of
LV-integration site profiles. In that regard,
biomolecules such as neurotrophic factors
represent novel candidates for enhancing
the expansion and survival of HSPCs, 19 while
the pyrimidoindole derivative UM171 wasrecently successfully employed for the ex vivo
expansion of LV transduced human mobilized
peripheral blood HSCs with long term
repopulation

capacity.18

The

therapeutic

outcomes can be further enhanced by
the integration of approaches aiming at
improving the homing and engraftmentof gene modified HSPCs. These strategies
include inhibition of CD2620 peptidase thatnegatively affects the levels of the chemokine
stromal cell-derived factor 1 (SDF-1) andpriming of HSCs with prostaglandin E2 prior
to transplantation.18,21,22

HSCGT: an efficient and safe
treatment for MNGIE?
Allogeneic HSCT for treatment of MNGIE
is associated with a high mortality rate andthe therapeutic outcomes are transient andlimited.23,24 As a result of the encouraging
therapeutic

outcomes

andincremental

improvements related to LV vector design,
HSCGT holds promise as a safe and effective
154

treatment for multiple life-threatening
diseases, including MNGIE.
Development of a LV vector-mediated
HSCGT protocol for the treatment of MNGIE
would require (i) a clinically relevant mousemodel, (ii) a clinically applicable LV vector,(iii) assessment of therapeutic outcomes,
beyond correction of the biochemical
phenotype, and (iv) assessment andminimizing the risks related to pre-transplantconditioning, transgene expression and
LV vectors.
The studies performed in this thesis
address these points, but clinical translation
requires additional studies to provide HSCGT
as a cure for MNGIE patients.
(i) The generation of Tymp-/-Upp1-/- mice
was instrumental to enhance the developmentof alternative treatments to cure MNGIE
patients.25 Tymp-/-Upp1-/- mice exhibita biochemical phenotype similar to MNGIE
patients (elevated dThd and dUrd), however,
only ≥18 month old mice demonstrate mild
neurological phenotypes. The lack of otherrelevant similarities to MNGIE such asalterations of mitochondrial DNA (mtDNA)
and pronounced motor dysfunctions, limits
the evaluation of the therapeutic outcomesbeyond the biochemical phenotype.26-28 To
enforce these phenotypes the nutritional
diet can be changed, consisting of increased
dThd and dUrd amounts, but mice still need
to be maintained for very long periods (up to
24 months).29 These limitations challenged
the suitability of Tymp-/-Upp1-/- mice to model
MNGIE. We therefore carefully characterized
the brain and intestinal phenotypes at an
earlier age to evaluate HSCGT efficacy. First,
we carefully examined the neurological aspectsof Tymp-/-Upp1-/- mice which confirmed
the previously reported white matter changes

General discussionin brain MRI and histology,25 but at an
earlier age, and additionally demonstrated
an abnormal phenotype of brain astrocytes.
Further experiments are required to elucidate
the nature of the cellular and molecular changes
observed in brain astrocytes morphology andthe altered mtDNA replication, and how that
relates to unbalanced nucleosides levels. In
this regard, experiments to evaluate ion-waterhomeostasis are relevant. Specifically, weobserved an increase in the thickness of
the astrocytes processes in Tymp-/-Upp1-/mice. This is similar to megalencephalicleukoencephalopathy with cysts (MLC)
mice models (Mlc1-null and Glialcam-nullmice). These studies provide evidence that
defective astrocytic fluid volume regulation
underlies the pathomechanism of MLC. 30,31
Future studies could be performed toinvestigate the status of volume regulatedanion channel (VRAC) currents andregulated volume decrease in astrocytesof Tymp-/-Upp1-/- mice. Additionally, we
report abnormalities in the myogenic andneurogenic compartments of the intestineof Tymp-/-Upp1-/- mice. However the mice
did not develop profound cachexia, unlike
MNGIE patients, perhaps due to ad libitum
feeding, or that the mice are double knockout for two genes unlike in MNGIE patients,or the physiological differences between
mice and man. Nonetheless, the intestinal
pathology of Tymp-/-Upp1-/- mice occurs at
an early age and is similar to the pathology
in MNGIE patients. Therefore, testing
potential treatments in the mouse model
may still predict the therapeutic response in
MNGIE patients.
(ii) Our results indicate that the developed
therapeutic LV vectors (PGK-TP(co) could
efficiently correct MNGIE phenotypes withthe following minor modifications: (a)The physiological promoter (hPGK) led tolower levels of TP expression which were
sufficient for phenotypic correction, without
the increased risk of transactivation of nearby
oncogenes as compared with the strong
promoter of the spleen focus forming virus(SF); (b) at low VCN/cell (median ≤ 2.3, MOI10); and (c) at moderate levels of engraftments(median, 77 %, PGK-TP(co)). Altogether,
these results indicate that the therapeutic
LV-PGK-TP and TPco vectors mediate
efficient correction, thereby abrogating
the necessity for further enhancement of
the vector performance. Although codon
optimization of TYMP did not enhance
TYMP transcription or protein levels, unlikein the case of other disease models,32,33 otheralgorithms might be investigated to still
improve protein production per VCN.
In order to control transgene expression,
enhance the biodistribution of- andimprove the reconstitution of- transduced
hematopoietic cells, numerous modifications
could be implemented. Cell specific targeted
transgene expression could be achieved
through the incorporation of cell specificpromoters or enhancers such as GP1BA,
ITGA2B, and PF4 megakaryocyte-specificgene promoters 34 to selectively drive moderatelevels of TYMP expression in platelets, a richsource of TP in healthy people. Tropism of LV
vectors can be altered through modificationsof LV envelopes to target long-term
repopulating stem cells, or the expression
restricted to certain cell types via miRNAde-targeting (reviewed in Goyvaerts C, etal. 2013).35 This would allow for strong andprolonged expression levels in target stem
cells, without the toxicity on nontarget cells.
Alternatively, targeted gene editing of HSCs

7

155

Chapter 7

with artificial endonucleases such as ZincFinger Nucleases (ZFNs), Transcription
Activator Like Effector Nucleases (TALENs)
and Clustered Regularly Interspaced Short
Palindromic Repeats/ CRISPR-associated(CRISPR/Cas) could reduce the risk ofinsertional oncogenesis through the direct
integration into pre-determined genomic loci.
In this regard, protocols are developed for
efficient gene targeting of HSCs by using ZFN,
augmented with adapted culture conditions.22
The delayed culture timing prior to genetargeting and the presence of StemRegenin
1 and prostagandin E2 to preserve stemness
permitted for efficient targeted integrationin human HSCs with long term repopulationin immunodeficient mice. The functionalityof this approach was proven by targeted
integration of the corrective cDNA into
IL2RG in HSCs from a SCID-X1 patient.
3-11% GFP+ were detected in the BM CD34+
subpopulations isolated from the patient aftertargeting for IL2RG correction. For MNGIE,
probably higher efficiency are required,since TYMP gene corrected cells, in contrast
with IL2RG corrected cells, might not confera selective advantage.
In contrast to ZFNs, TALENS targetingthe same site of CCR5 were reported tohave a reduced off-target activity.36 TALENs
were successfully used for targetingthe human β-globin locus; i.e. correction of
the pathological mutations in iPSCs frompatients of β-thalassemia37 and sickle cellanemia,38 to express normal b-globin in
the differentiated hematopoietic progenitorsand erythroblast. This is probably also
feasible for correction of TYMP as well.

The CRISPR/Cas9 system can be used for
efficient gene editing of primary murine andhuman HSPCs,39 or patient-derived induced
pluripotent stem cells (iPSCs). In this approach,
CRISPR/Cas9 is applied for correction of
the disease underlying mutation in patientderived iPSCs, which are differentiatedafterwards to, for instance, hematopoietic
progenitor cells, for transplantation inpatients. The majority of lysosomal storage
diseases are candidate targets for treatment
with this approach, because of the monogenic
mutations and the ability of gene corrected
cells to excrete the deficient enzyme.40 Theseadvancements and progress towards clinical
trials (e.g. treatment of HIV infection1, andmucopolysaccharidosis I and II2) rendertherapeutic gene editing an attractive
treatment option, feasible for application in
MNGIE patients as well. However, several
limitations need to be considered prior
to the clinical application of iPSCs. These
include the large-scale manufacturing steps
such as the genetic re-programming andin vitro expansion, increased incidence ofdeletions or changes in DNA copy number
and thereby tumorigenesis, which is also
underlined by the use of integrating viruses
and oncogenes, and risks of immune response
or graft rejection. Furthermore, translationof gene editing strategies into clinicalsettings requires further enhancements ofthe delivery, efficiency and specificity of
the gene correction machinery. First, invivo gene editing is usually performed with
AAV,41 which imposes challenges including
off-target editing related to the constitutively
expressed nucleases, and immunity against

https://clinicaltrials.gov/ (2017) 1(NCT01252641), 2 (NCT02702115, NCT03041324)

156

General discussion

certain serotypes. On the other hand, for
ex vivo gene targeting of HSCs, transient
expression of the nuclease is sufficient and canbe achieved through electroporation or nonintegrating LV vectors.22,39 In order to avoid
excessive expression of nucleases and offtarget editing integrating LV vectors are notpreferred. Second, the efficiency of targeted
gene correction could be enhanced bymodulation of double strand break pathways,
which are dependent on the target cell cycle.
Modulation of ex vivo cell culture conditions
could be useful to control the cell cycle.22
In addition, several approaches can beapplied in order to achieve above normal
TYMP

expression

levels(as

discussed

in iii) including the targeted integrationof an expression cassette with a strong
promoter in a pre-selected safe harbor such
as AAVS1, or fusion of the open reading
frame of TYMP with the mRNA of a highly
expressed endogenous gene such as CD45
in HSPCs. As in the case for the treatmentof hemophilia B, in vivo genome editing byusing ZFNs and AAV2/6 was successfully
used for the insertion of a therapeutic copyof human factor 9 into the albumin locus inliver hepatocytes, leading to the production oftherapeutic levels of human factor 9 (>1% ofnormal) in mice and non-human primates.42
Finally,

off-target

gene

editing

andconstitutively expressed nucleases could resultin undesired alterations of cancer-related
genes or lead to loss of function, therefore
the specificity of CRISPR/Cas9 targeting canbe enhanced by selection of the nucleases
used,43 or by using truncated guide RNA,
guide

RNA

extension,

Cas9

RNA-guided

FokI–dCas9

nucleases,

engineered Cas9. 44

nickases,or(iii) Above-normal TP activity was
required for restoration of neurological andintestinal phenotypes and was achieved
with LV-PGK-TP(co) at a moderate LV dose
(MOI 10). This is similar to findings in othermetabolic disorders, where above-normal
expression of the therapeutic enzyme led tocorrection of disease phenotypes which areotherwise not responsive to traditional HSCT.
This includes the amelioration of neurologicalphenotypes in mucopolysaccharidose II 45 andgloboid cell leukodystrophy mice46 (reviewed
in Biffi A, et al. 2017).47 Only the highest doseused in our studies in Tymp-/-Upp1-/- mice,an MOI of 10, resulted in above-normal
levels of TP activity in blood (range, 68-188
nmol/h/mg protein), was just sufficient at
normalizing intestinal TP activity and rescued
the pathology after HSCGT, while HSCT in
MNGIE patients did not rescue the intestinal
pathology despite biochemical correction
as demonstrated in Chapters 4 and 5 andby Halter et al. (range, 262-1285 nmol/h/mg
protein).23 Graft versus host disease or graft
rejection following allogeneic HSCT, timingof treatment (HSCGT was administered at
the same age when the intestinal pathology
was minor, versus MNGIE patients who areusually treated at a late stage of the disease),or interspecies differences could also explain
the contrasting responses of human and miceto treatments.
In contrast to using LV-mediated HSCGT,
above-normal TP levels in blood were notachieved through AAV2/8-mediated liver
directed gene therapy in Tymp-/-Upp1-/- mice,even with high vector dose (>2×1011 vg/kg).
Importantly, nucleoside accumulation was
not reduced in the intestine of these mice.27
AAV gene therapy for MNGIE is an attractive
treatment option because it does not require

7157

Chapter 7

pre-conditioning or a suitable HSCs donor.
However, compared with clinical trials forhemophilia B, in which a small proportionof transduced hepatocytes is sufficient toconvert a severe patient into a moderate
phenotype, it is speculated that an effective
treatment for MNGIE would require an
improved expression cassette, targeted
expression in affected organs or a higher AAV
vector dose. A higher dose increases the riskof eliciting immunity towards the viral capsid
and liver toxicity. The pros and cons of AAVand LV-based gene therapy approaches are
discussed in more detail in Chapter 2.
In addition to the above-normal TP
levels, the timing of treatment is critical forthe resolution of the intestinal phenotypes.
HSCGT was administered prior to detectable
pathological appearance (at 2 months ofage), whereas MNGIE patients receive
HSCT usually when symptomatic (i.e. after
development of a clinical phenotype). To that
end, early diagnosis through genetic screeningof newborns is critical. Nonetheless, vector
and transplanted gene modified cell dose
escalation experiments involving a large groupof mice is required to establish the minimaldose sufficient for phenotypic correction
and without cellular or molecular toxicity
before translation into a clinical protocol fortreatment of MNGIE patients.
Moreover,albeitcorrectionofthe biochemical, neurological, and intestinal
phenotypes we were unable to evaluate
the contribution of HSCGT to recovery ofapparent clinical phenotypes such as motor
function and cachexia, due to their absencein the Tymp-/-Upp1-/- mice. Tymp-/-Upp1-/- mice
fed with a diet consisting of dThd and dUrdin order to induce the clinical phenotypes 29
could be used to assess the effects of HSCGT
158

on recovery of apparent phenotypes suchas motor dysfunction. (iv) Adverse events
could occur that are related to toxicity ofthe pre-transplant conditioning, transgene
over expression or immunity against
the transgene product, or genotoxicity ofintegrated LV vectors. In this thesis, weaddressed these points in relation to HSCGT
for MNGIE. Prior to HSCT, a relatively mild
conditioning consisting of busulfan andfludarabine or in combination with anti T
cell antibodies, is usually applied. 23 However,an alternative mild conditioning could be
preferred for treatment of MNGIE patients
who are usually in a very poor condition.
Novel
non-cytoreductive
conditioning
approaches have been successful in preclinicalstudies, including the administration of
HSC mobilizing cytokines such as humangranulocyte colony stimulating factor,48 orantagonists of endogenous HSC markers suchas anti c-kit 49 or anti CD45.50 Our results
show that following sub-lethal total body
irradiation of Tymp-/-Upp1-/- mice, moderatelevels of engraftment were sufficient for
disease correction. Therefore, application of
the novel non-cytoreductive pre-transplantconditioning may be adequate to achievesufficient levels of donor cell chimerism fordisease correction, and avoid toxicity andfurther deterioration of mtDNA, in particularwhen combined with the advancementsof gene transfer protocols and the ex vivo
expansion. In order to assess any potential
phenotoxicity related to TYMP overexpression, further in depth experiments
are required. In this regard, it is importantto evaluate if HSCGT sufficiently restoresthe depleted dCTP levels and investigate
any potential negative effects of TP overexpression on dNTPs pool homeostasis,

General discussion

mtDNA replication, cell cycle arrest,
and apoptosis.51,52
The results of this thesis indicate that
HSCGT for MNGIE can be performed
with low risks of in vivo mutagenesis or
integration nearby oncogenes. Follow up
of primary and secondary recipients of LVPGK-TP(co) for 22 months revealed no
detectable hematological abnormalities andthe LAM-PCR demonstrated benign vector
integration patterns. No differences in
the frequency of integration near oncogenes
were observed between the control and genetherapy treated groups, or classified basedon the promoter (PGK vs. SF) or transgene(therapeutic vs. GFP) used and were alsosimilar to those obtained for different
inherited diseases such as in Il2rg-/- andGaa-/- mice. Two secondary recipients in
the PGK-TP-GFP treatment group developed
B cell lymphoma; the clone contained a single
dominant integration site in gene Zfp207.
Zfp207 is required for proper chromosomealignment53 and therefore, interruption
by for instance, a LV insertion could haveaugmented oncogenesis. This requires further
experiments to elucidate the role of Zfp 207 asa potential tumor related gene. For example,
DNA and RNA sequencing can be performed

in order to profile altered signaling pathways
or changes in the mitotic checkpoint protein
Budding Uninhibited by Benzimidazoles 3(BUB3), the main target for Zfp207, as well
as functional studies that entitle knockdownof Zfp207 in vitro or ex vivo to determine
the effect on cycling HSCs, and to determine
its role in the contribution of oncogenesis.
In conclusion, LV vector-mediated
HSCGT for MNGIE patients is a feasible
treatment option which can successfully
reverse the biochemical, neurological, andintestinal phenotypes in Tymp-/-Upp1-/mice without apparent toxicity. Clinicalapplication of HSCGT for the treatment
of MNGIE patients would require further
experiments to assess the minimal LV dose
for the correction of clinical phenotypes
such as motor function or cachexia, andadditional evaluation of potential toxicity
related to TYMP over expression. Futureadvancements could involve strategies todirect and control transgene expression intarget cells, and strategies for improving
the HSCGT procedure such as enhanced LV
transduction of HSPCs and ex vivo expansion
and selection of gene modified cells prior totransplantation, in addition to the applicationof non-cytoreductive preconditioning.

7

159

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Appendix

Summary
Samenvatting
Arabic summary (‫)ملخص‬List of terms and abbreviations
PhD portfolio
List of publications
Curriculum vitae
Acknowledgements

Summary| |SUMMARY
Hematopoietic stem cell gene therapy(HSCGT) is an attractive treatment option
for a wide range of disorders. The initialclinical trials for X- linked severe combined
immunodeficiency(SCID-X1)
and
ADA-SCID applied γ-RV vectors successfully,
but hematological malignancies occurredin SCID-X1 due to vector integrationand transactivation of nearby oncogenes.
Therefore, efforts were directed towards
finding alternative viral vector systems
leading, eventually, to the developmentof the new 3rd generation self-inactivating(SIN) lentiviral (LV) vectors with enhanced
efficiency and benign integration profiles.
Mitochondrial
neurogastrointestinal
encephalomyopathy (MNGIE) is currently
treated with allogeneic hematopoietic stem
cell transplantation (allogeneic HSCT).
Allogeneic HSCT successfully reverses
the biochemical phenotype. However, HSCT
is associated with high mortality rates,
requires the availability of matched donor
hematopoietic stem and progenitor cells(HSPCs) while knowledge about the longterm therapeutic outcome is limited. LVmediated HSCGT uses autologous cells, may
require lower pre-conditioning regimens andis expected to provide long-term systemic
disease correction as discussed in Chapter 2.
In Chapter 3, we optimized the ex vivo
culture conditions of LV vector mediated genetransfer into HSPCs to achieve efficient genemarking within a short transduction time
period, with a minimal number of integrated
vector copies, and minimal cytokine additions.
The results indicate the feasibility to improvegene transfer by increasing the density ofmouse, rhesus, or human HSPCs target

cells. Additionally, overnight transductionof mouse and human HSPCs in stem cell
medium with addition of thrombopoietin
(TPO)
only
enhancedtransduction
sufficiently for in vivo purposes. The additionof the growth factors SCF, TPO, Flt3-L
at relatively low concentrations provided
the most efficient gene transfer (> 60% GFP
marking) while maintaining progenitor andstem cells repopulation capacity. Altogether,
these findings open intriguing possibilities forovercoming the limitations of the current genetransfer protocols: an enhanced efficiency andimproved safety of gene correction, minimal
ex vivo manipulation during a short culture
period, and the added economic value of using
small amounts of clinical grade lentiviral
vectors and cytokines.
Chapters 4 and 5 focus on the evaluationof the efficiency and safety of LV vectormediated HSCGT as a treatment for MNGIE
in Tymp-/-Upp1-/- mice. Therapeuticallyapplicable SIN LV vectors were used
containing
thephysiologicalhuman
phosphoglycerate kinase (hPGK) promoter
for ubiquitous expression of native or codon
optimized human TYMP cDNA (PGK-TPor PGK-TPco, respectively). LV-SF-TPcobearing the strong spleen focus forming
virus (SF) promoter was used for safety
evaluation to highly express TP, and to assess
potential genotoxicity. The results in Chapter
4 demonstrated persistent TP activity andclearance of nucleosides in the brain of treatedTymp-/-Upp1-/- mice at relatively low vector
copy numbers (PGK-TP(co): MOI 10, range;
0.2-3.6 VCN/cell). Here, we showed that thisresulted also in reversal of the phenotypicalterations apparent in brain astrocytes ofA

167

Appendix

Tymp-/-Upp1-/- mice, as well as reduction ofwhite matter edema demonstrated by brain
MRI and immunohistological analysis. Long
term follow up of recipients of HSCGT
revealed no side effects from the procedure:(i) Graft failure or toxicity related to sub-lethal
total body irradiation was not observed. (ii)
Survival curves of mice in the PGK-TP(co)treatment groups did not differ significantly
from controls. (iii) LV- related insertionalmutagenesis or hematopoietic transformation
were not observed after a total of 22 months
follow up of primary and secondary recipientsof HSCs transduced by therapeutic LV vectors
PGK-TP(co). (iv) LAM-PCR and sequencing
demonstrated a polyclonal integration pattern
and no bias towards integration near or
selection of proto-oncogenes.
Gastrointestinal
manifestations
areprominent and the main cause of deathamong MNGIE patients. In Chapter 5 we
examined the pathology of the small intestineof MNGIE patients and Tymp-/-Upp1-/mice and the impact of allogeneic HSCT
or gene therapy, respectively. Our findings
confirmed the previously reported atrophyof the muscularis propria and absence ofinterstitial cells of Cajal in MNGIE patients.
HSCT did not resolve these pathologicalcharacteristics in the short period
studied post transplantation. This might
explain the repeatedly reported persistent
gastrointestinal manifestations after treatment.
In contrast, HSCGT rescued the atrophic
muscularis propria of the small intestine intreated Tymp-/-Upp1-/- mice 10 months after
treatment. This suggests that the myogenic
changes can be reversed when gene therapy

168

is provided early in life. Finally, in Chapter 6:
we further evaluated in depth the LV vectors
integration profiles in gene modified human,rhesus or murine HSCs cultured in vitro, andin vivo after transplantation in disease modelsof SCID, Pompe, MNGIE and healthy mice.
The integration patterns were similar in all
species studied, and were not skewed based on
the type of promoter (SF versus PGK, RAG1p,
TCRβp, UCOE and γcPr), or transgene used (GFP versus Il2rg, Rag1, Rag 2, Gaa and Tymp).
In conclusion, the results presented in thisthesis demonstrate efficient and persistentcorrection of the biochemical phenotype
in affected organs of Tymp-/-Upp1-/- mice,brain neurological phenotypes (whitematter edema and astrocytes morphology),
and intestinal myopathy (atrophy ofthe muscularis propria), at relatively low
LV-PGK-TP(co) vector copy number percell and without obvious vector or disease
related phenotoxicity or genotoxicity. Due tothe lack of some of the major neurological or
gastrointestinal clinical phenotypes in Tymp-/Upp1-/- mice (such as motor dysfunction orcachexia) we should be cautious in translating
our findings from mice to MNGIE patients.
Nonetheless, it is recommended to perform
more studies before clinical application,
including evaluating HSCGT in moreclinically relevant models that mimic human
MNGIE disease (e.g. Tymp-/-Upp1-/- mice fed
with a diet to enforce the phenotypes), dosingof LV vector and transplanted gene modified
cells, and more thorough assessment of vector
biodistribution and safety studies addressing
potential phenotoxicity and genotoxicity
in depth.

Samenvatting (Dutch summary)| |Samenvatting (Dutch summary)
Hematopoietische
stamcelgentherapie(HSCGT)iseenaantrekkelijkebehandelingsoptie voor een breed scalavan aandoeningen. Bij de eerste klinischestudies naar X-linked ernstig gecombineerde
immunodeficiëntie (SCID-X1) en adenosine
deaminase (ADA)-SCID heeft men γ-RV
vectoren met succes toegepast, maar bij
SCID-X1
ontstonden
hematologischemaligniteiten als gevolg van vectorintegratie
en transactivatie van nabije oncogenen.
Daarom werd gezocht naar alternatieve
virale vectorsystemen. Dit leidde uiteindelijk
tot de ontwikkeling van de nieuwe, derde
generatie zelf-inactiverende (SIN) lentivirale(LV) vectoren met verbeterde efficiëntie en
goedaardige integratieprofielen.
Mitochondriële
neurogastrointestinale
encefalomyopathie(MNGIE)
wordt
momenteelbehandeldmetallogenehematopoietische
stamceltransplantatie(allogene HSCT). Deze therapie leidt tot
reversie van het biochemische fenotype, maar
is geassocieerd met hoge sterftecijfers. Verderdienen matchted donor hematopoietischestamenvoorlopercellen(HSPCs)beschikbaar te zijn, en er is nog weinig
bekend over de uitkomst op de lange termijn.
LV-gemedieerde HSCGT maakt gebruik van
autologe cellen, kan mogelijk toe met lagere
pre-conditioneringsregimes en kan naar
verwachting systemische ziekte op termijn
corrigeren, zoals besproken in Hoofdstuk 2.
In Hoofdstuk 3 optimaliseerden wede ex vivo kweekomstandigheden van
LV-vector gemedieerde genoverdracht inhematopoietische stam en voorloper cellen(HSVC’s). Het doel hiervan was om efficiënte
transductie te bereiken binnen een korte

incubatietijd, met een minimaal aantalgeïntegreerde vectorkopieën en minimale
cytokine toevoegingen. De resultaten geven
aan dat het haalbaar is de genoverdracht teverbeteren door het verhogen van de dichtheidvan muizen-, rhesus- of humane HSVC-cellen.
Ook zagen we dat de overnacht transductie van
muis- en humane HSVC’s in stamcelmedium
waaraan alleen trombopoëtine (TPO)
was toegevoegd als cytokine, significant
verbeterde transductie opleverde voor invivo doeleinden. Toevoeging van relatieflage concentraties van de groeifactoren
SCF, TPO, Flt3-L gaf de meest efficiënte
genoverdracht (60% GFP-markering) met
behoud van de mogelijkheid tot repopulatie
van voorloper- en stamcellen. Alles bij
elkaar genomen bieden deze bevindingenmogelijkheden om de beperkingen vande huidige genoverdrachtprotocollen teverbeteren door een verhoogde efficiëntie enminder veiligheidsrisico’s m.b.t. insertionele
mutagenese, met minimale ex vivo manipulatie
gedurende een korte kweekperiode en
de toegevoegde commerciële waarde van het
gebruik van kleine hoeveelheden klinische
lentivirale vectoren en cytokines.
In de Hoofdstukken 4 en 5 lag de nadruk
op de evaluatie van de efficiëntie en veiligheid
van LV vector-gemedieerde hematopoietischestam en voorloper cel gentherapie (HSVGT)
als een behandeling voor MNGIE in Tymp -/Upp1 -/- muizen. Hierbij werden therapeutisch
toepasbare SIN LV-vectoren gebruikt met defysiologische humane fosfoglyceraatkinase(hPGK) promotor om algemene expressie vanoorspronkelijk- of codon-geoptimaliseerd
humaan TYMP cDNA te bewerkstelligen(respectievelijk PGK-TP of PGK-TPco).

A

169

Appendix

Voor evaluatie van de veiligheid werd de
LV-SF-TPco gebruikt met de sterke miltfocusvormende viruspromotor waarbij een
hoge TP-expressie wordt verkregen om
de mogelijke genotoxiciteit te beoordelen.
De resultaten in Hoofdstuk 4 wezen op
persisterende TP-activiteit en klaring van
nucleosiden in de hersenen van behandeldeTymp-/- Upp1-/- muizen bij relatief lage
aantallen vector-kopieën (PGK-TP(co): MOI
10, bereik, 0,2-3,6 VCN /cel). Dit resulteerde
niet alleen in de correctie van de fenotypischeveranderingen die te zien waren inde hersenastrocyten van Tymp-/- Upp1-/muizen, maar ook in vermindering vanoedeem in de witte stof, zoals aangetoond
met hersen-MRI en immunohistochemie.
Bij de lange termijn follow-up van de vanbehandelde HSCGT muizen zijn geen
nadelige bijwerkingen van de procedure
gezien: (i) Falen van donormateriaal of
toxiciteit door de subletale bestraling van
het gehele lichaam zijn niet waargenomen.
(ii) De overlevingscurven van de muizen inde PGK-TP(co) behandelingsgroepen waren
statistisch niet significant verschillend van
de overlevingscurven van de controledieren.
(iii) LV-gerelateerde insertiemutageneseof hematopoietische transformatie werden
niet waargenomen na in totaal 22 maanden
follow-up van de primaire en secundaire
ontvangers van door therapeutische LVvectoren (PGK-TP(co)) getransduceerde
HSC’s. (iv) LAM-PCR en sequencing lieten
een polyclonaal integratiepatroon zien zonder
bias naar integratie in de nabijheid van of in
proto-oncogenen.
Gastrointestinale aandoeningen komen
veel voor en zijn de belangrijkste doodsoorzaak
bij MNGIE-patiënten. In Hoofdstuk 5 hebben
we onderzoek gedaan naar de pathologie van
170

de dunne darm van MNGIE-patiënten en
Tymp-/- Upp1-/- muizen en de effecten van
respectievelijk allogene HSCT of gentherapie.
Onze bevindingen bevestigden de al eerder
gerapporteerde atrofie van de muscularis
propria en afwezigheid van interstitiële
cellen van Cajal bij MNGIE-patiënten.
HSCT liet geen effect zien in de korte tijd
tot enkele maanden na transplantatie. Dit
kan de herhaaldelijk gemelde aanhoudende
gastrointestinale klachten na de behandelingverklaren. Daarentegen herstelde HSCGTde atrofische muscularis propria van de dunne
darm in behandelde Tymp-/- Upp1-/- muizen 10
maanden na de behandeling. Dit suggereert
dat de myogene veranderingen omgekeerd
kunnen worden wanneer gentherapie vroeg
in het leven wordt gegeven. Tenslotte,
Hoofdstuk 6 betreft een diepgaandere
evaluatie van de LV vector integratieprofielen
in gen-gemodificeerde humane, rhesus ofmuizen HSVC’s, gekweekt in vitro en in vivo
na transplantatie in ziektemodellen van SCID,
Pompe, MNGIE en in gezonde muizen. De
integratiepatronen waren vergelijkbaar in alleonderzochte species en niet afhankelijk van
het type promotor (SF versus PGK, RAG1p,
TCRβp, UCOE en γcPr) of het gebruikte
transgen (GFP versus IL2RG, RAG-1, Rag 2,
Gaa en Tymp).
Concluderend blijkt uit de resultaten in
dit proefschrift een effectieve en aanhoudendecorrectie van het biochemische fenotype
in aangedane organen van Tymp-/- Upp1-/muizen, hersen- fenotypes (witte stof oedeem
en astrocyten morfologie) en intestinale
myopathie (atrofie van de muscularis propria),
bij een relatief aantal LV-PGK-TP(co)vector copien per cel en zonder duidelijke
vector- of ziekte gerelateerde fenotoxiciteit ofgenotoxiciteit. Door het ontbreken van enkele

Samenvatting (Dutch summary)belangrijke neurologische of gastrointestinale
klinische fenotypes in Tymp-/- Upp1-/- muizen(zoals motor dysfunctie of magerzucht)
moeten we terughoudend zijn bij het vertalen
van onze bevindingen bij de muizen naar
MNGIE-patiënten. Het is aan te bevelen meer
onderzoek te doen voordat dit klinisch kan
worden toegepast. Zoals het evalueren van
HSCGT in meer klinisch relevante modellen

van humane MNGIE-muizen (bijvoorbeeld
Tymp-/- Upp1-/- muizen die dieetvoeding krijgenom de fenotypen te forceren), de doseringvan LV vector en getransplanteerde gengemodificeerde cellen, en verdere diepgaande
evaluatie van de biodistributie van vectors
alsmede grondiger veiligheidsstudies naar
de mogelijke fenotoxiciteit en genotoxiciteit.

A

171

‫‪Appendix‬‬‫سالمة زي ــادة تركيز بروتين فوسفوريليز ثايميدين )‪،(TP‬‬‫فئة ‪ ،Tymp-/-Upp1-/-‬إضافة إلى دراسة أثر زراعة نخاع‬‫ولتقييم مدى إمكانية وجود سمية ورائية‪ .‬تشير النتائج الواردة‬‫العظم بالخاليا الجذعية )‪ (allogeneic HSCT‬أو العالج‬‫فــي الفصل الرابع إلــى وجــود نشاط متواصل لبروتين ‪TP‬‬‫الجيني )‪ ،(gene therapy‬على التوالي‪ .‬أكدت النتائج التي‬‫واختفاء النيوكليوسيدات في دماغ الفئران ‪Tymp-/-Upp1-/-‬‬‫توصلنا إليها االدعاءات السابقة بحدوث ضمور في العضلة‬‫التي خضعت للمعالجة‪ ،‬وذلــك بعدد نسخ منخفضة نسبياً‬‫الخصوصة )‪(atrophy of the muscularis propria‬‬‫()‪ ،MOI 10 :PGK-TP(co‬المدى‪VCN 3.6-0.2 :‬‬‫وعدم وجود أي خاليا خاللية من نوع كاجال )‪ (Cajal‬لدى‬‫لكل خلية)‪ .‬هنا‪ ،‬قمنا بإظهار كيف أن هــذا تسبب أيضاً‬‫المصابين بمرض منجي )‪ .(MNGIE‬لم يتسبب العالج‬‫فــي عـكــس الـتـغـيـرات الـمـظـهـريــة ال ـتــي حـصـلــت فــي الـخــايــا‬‫بــال ـخــايــا ال ـجــذع ـيــة )‪ (HSCT‬في ح ــل ه ــذه الـخـصــائــص‬‫النجمية )‪ (astrocytes‬لــدمــاغ الـفـئـران مــن فـئــة ‪Tymp-‬‬‫المرضية خــال الفترة القصيرة التي تم دراستها بعد إجـراء‬‫‪ /-Upp1-/-‬الـتــي خضعت للمعالجة‪ ،‬كما أنـهــا تسببت في‬‫عملية ال ــز ارع ــة‪ .‬يمكن لـهــذه النتيجة أن تفسر التغييرات‬‫تقليل وذمــة الـمــادة البيضاء )‪(white matter edema‬‬‫الظاهرة في الجهاز الهضمي التي يتم اإلبالغ عنها بشكل‬‫التي ظهرت في صــورة الرنين المغناطيسي )‪ (MRI‬التي‬‫متكرر لدى المرضى بعد تلقي العالج‪ .‬وعلى عكس ذلك‪،‬‬‫أجـريــت لـلــدمــاغ وتـحــالـيــل الكيمياء الهيستولوجية المناعية‬‫فإن العالج الجيني للخاليا الجذعية الدموية )‪(HSCGT‬‬‫)‪ .(immunohistological analysis‬أظهرت المتابعة‬‫تسببت في تقليل ضمور العضالت المخصوصة في األمعاء‬‫طــويـلــة األم ــد بـعــد تـلـقــي ال ـعــاج الـجـيـنــي لـلـخــايــا الـجــذعـيــة‬‫الدقيقة لدى الفئران من فئة ‪ Tymp Upp1‬بعد ‪ 10‬أشهر‬‫ال ــدم ــوي ــة )‪ (HSCGT‬عـ ــدم وج ـ ــود أي أع ـ ـ ـراض جــانـبـيــة‬‫من تلقي العالج‪ .‬قد تشير هذه النتائج إلى أنن يمكن عكس‬‫للعملية‪( :‬أ) لــم يـتــم مــاحـظــة فـشــل ال ــز ارع ــة أو أي تسمم‬‫الـتـغـيـرات الناشئة مــن العضل فــي حــال تــم إعـطــاء العالج‬‫ناتج عن تعرض الجسم ألشعة سينية شبه قاتلة‪( .‬ب) لم‬‫الجيني فــي مرحلة مبكرة مــن الـحـيــاة‪ .‬وأخ ـي ـ اًر‪ ،‬فــي الفصل‬‫تختلف منحنيات البقاء على قيد الحياة بشكل ملحوظ عند‬‫السادس‪ ،‬قمنا بــإجـراء تقييمات أكثر عمقاً لمكونات الــدم‬‫الفئران التي تمت معالجتها باستخدام )‪ PGK-TP(co‬عن‬‫مرتبطة بالفيروسات البطيئة )‪ (LV‬لدى الكائنات التي طبقت‬‫المدى الطبيعي‪( .‬ج) لم يتم مالحظة أي طفرات إدراجية أو‬‫عليها تعديالت جينية مــن البشر‪ ،‬أو الـقــردة الصغيرة‪ ،‬أو‬‫تغيرات في مكونات الدم مرتبطة بالفيروسات البطيئة )‪(LV‬‬‫الخاليا الجذعية المكونة للدم من الفئران مستنبتة في البيئة‬‫بـعــد م ــدة وصـلــت إل ــى ‪ 22‬ش ـه ـ اًر مــن الـمـتــابـعــة للمستقبلين‬‫المخبرية وفي الوسط الحيوي بعد عملية الزراعة في الفئران‬‫األول ـي ـي ــن وال ـثــانــوي ـيــن ل ـل ـخــايــا ال ـجــذع ـيــة ال ـتــي ت ــم تـحــويـلـهــا‬‫المصابة بمرض العوز المناعي المكتسب الشديد )‪،(SCID‬‬‫باستخدام الفايروس البطيء العالجي )‪( .PGK-TP(co‬د)‬‫داء اختزان الغاليكوجين النمط الثاني (‪ ،)Pompe‬ومرض‬‫أظهرت تحليالت ‪ LAM-PCR‬وتحليالت التسلسل وجود‬‫منجي )‪ ،(MNGIE‬إضافة إلى فئران سليمة‪ .‬كانت أنماط‬‫نمط اندماج متعدد النسائل من دون أي انحياز نحو حدوث‬‫االندماج متشابهة لدى جميع األصناف التي تم دراستها‪ ،‬ولم‬‫اندماج بالقرب من أو في أي تجمع لطليعة الجين الورمي‪.‬‬‫يظهر أي اختالف بناءاً على نوع المحفز المستخدم (‪SF,‬‬‫تـعــد مـظــاهــر األل ــم فــي الـجـهــاز الـهـضـمــي ســائــدة وتعد‬‫‪ ،)PGK, RAG1p, TCRβp, UCOE, γcPr‬أو الجين‬‫الـسـبــب الـرئـيـســي لـلــوفــاة ل ــدى الـمــرضــى الـمـصــابـيــن بمرض‬‫المستخدم (‪.)GFP, Il2rg, Rag1, Rag 2, Gaa, Tymp‬‬‫مـنـجــي )‪ .(MNGIE‬فــي الفصل الخامس قـمـنــا بفحص‬‫في الختام‪ ،‬فإن النتائج الـواردة في هذه الرسالة تظهر‬‫وتحليل األمراض الظاهرة في األمعاء الدقيقة لدى المرضى‬‫تصحيح فـعــال ومستمر للنمط الـظــاهــري البيوكيميائي في‬‫المصابين بـمــرض منجي )‪ (MNGIE‬ول ــدى الـفـئـران من‬‫األع ـض ــاء الـمـصــابــة لـلـفـئـران مــن فـئــة ‪،Tymp-/-Upp1-/-‬‬‫‪-/-‬‬‫‪-/-‬‬‫‪172‬‬

‫)‪ (Arabic summary‬ملخص‬‫|)‪ (Arabic summary‬ملخص‬‫يعد الـعــاج الجيني بالخاليا الجذعية )‪ (HSCGT‬خيار‬‫الفايروسية‪ ،‬والحد األدنى من إضافات السيتوكينات‪ .‬تشير‬‫عــاجــي مغر للعديد مــن االضـطـ اربــات المرضية‪ .‬أظهرت‬‫النتائج إلــى فاعلية تحسين نقل الجينات مــن خــال زيــادة‬‫نتائج التجارب السريرية األولية لمرضى داء نقص المناعة‬‫كثافة الخاليا الجذعية و السلفية المكونة للدم المستهدفة‬‫المشترك الشديد )‪ (SCID-X1‬و )‪ (ADA-SCID‬بأنها‬‫المستخلصة مــن الـفـئـران‪ ،‬أو الـقــرد الصغير‪ ،‬أو االنـســان‪.‬‬‫تتوافق بنجاح مع النواقل الفايروسية القهقرية من نوع جاما‬‫ع ــاوة عـلــى ذل ــك‪ ،‬فــإن عملية نـقــل الـجـيـنــات الـتــي تـتــم بين‬‫)‪ ،(γ-RV vectors‬إال أن األورام الدموية الخبيثة حصلت‬‫ليلة وضحاها للخاليا الجذعية والسلفية المكونة للدم لدى‬‫فــي حــالــة نـقــص الـمـنــاعــة الـمـشـتــرك الـشــديــد )‪(SCID-X1‬‬‫الفئران واإلنـســان في مستنبت الخاليا الجذعية مع إضافة‬‫بسبب اندماج الفايروسات القهقرية وعملية التفعيل النسخي‬‫ثرومبوبويتين )‪ (TPO‬فقط تسببت في تحسين عملية نقل‬‫للجينات المسرطنة المجاورة‪ .‬وعليه‪ ،‬تم تركيز الجهود نحو‬‫ال ـمــادة الــو ارثـيــة ألغ ـراض الــد ارســة فــي البيئة الــداخـلـيــة ‪(in‬‬‫أدت‪ ،‬في نهاية المطاف‪ ،‬إلى‬‫إيجاد أنظمة فايروسية بديلة ّ‬‫)‪ .vivo‬إن إض ــاف ــة ع ـوام ــل ال ـن ـمــو ‪ ،SCF‬و ‪ ،TPO‬و‬‫تطوير الجيل الثالث من الفايروسات البطيئة )‪ (LV‬ذات‬‫‪A‬‬‫التعطيل التلقائي )‪ (SIN‬ذات الكفاءة العالية والتي تتمتع‬‫بالكفاءة األكثر(بنسبة تفوق الـ ‪ %60‬على مؤشر ‪،)GFP‬‬‫بفاعلية محسنة وبروفايالت إندماج حميدة‪.‬‬‫مع الحفاظ على قدرة إعادة إنتاج الخاليا الجذعية والخاليا‬‫في الوقت الحالي‪ ،‬يتم عالج مرض منجي )‪(MNGIE‬‬‫السلفية‪ .‬في اإلجمال‪ ،‬تساعد هذه النتائج في فتح احتماالت‬‫ع ـ ــن ط ـ ـريـ ــق ز ارع ـ ـ ـ ــة ن ـ ـخـ ــاع الـ ـعـ ـظ ــم ب ــالـ ـخ ــاي ــا ال ـج ــذع ـي ــة‬‫مثيرة لالهتمام لتخطي القيود التي تنتج عــن بــروتــوكـوالت‬‫)‪ .(allogeneic HSCT‬تنجح عملية زراعة نخاع العظم‬‫نقل الجينات الحالية‪ :‬كفاءة أعلى وتحسين في مدى سالمة‬‫بالخاليا الجذعية بعكس النمط الظاهري البيوكيميائي‪ .‬إال‬‫التصحيح الجيني‪ ،‬إضافة إلى الحد األدنــى من التعديالت‬‫أن عمليات ز ارع ــة نـخــاع العظم بالخاليا الجذعية ترتبط‬‫عـلــى الـبـيـئــة الـخــارجـيــة خ ــال ف ـت ـرة تنبيت قـصـيـرة‪ ،‬والقيمة‬‫بـنـســب وف ـيــات عــال ـيــة‪ ،‬وه ــي تـتـطـلــب وج ــود مـتـبــرع تتطابق‬‫االق ـت ـصــاديــة الـمـضــافــة ال ـتــي نـحـصــل عـلـيـهــا عـنــد اسـتـخــدام‬‫خالياه الجذعية وخالياه السلفية )‪ (HSPCs‬مع المريض‪،‬‬‫كميات صغيرة من الفايروسات البطيئة والسيتوكينات‪.‬‬‫كما أن المعرفة بالنتائج العالجية طويلة األمد محدودة‪ .‬يتم‬‫يتم التركيز في الفصل الرابع والفصل الخامس على‬‫خالل العالج الجيني للخاليا الجذعية الدموية )‪(HSCGT‬‬‫تـقـيـيــم م ــدى ك ـفــاءة ودرجـ ــة أم ــان ال ـفــايــروســات الـبـطـيـئــة في‬‫بــاس ـت ـخــدام ال ـف ــاي ــروس ــات الـبـطـيـئــة اس ـت ـخــدام ال ـخــايــة ذات ـيــة‬‫الـعــاج الجيني للخاليا الجذعية الــدمــويــة ‪(LV vector-‬‬‫المنشأ‪ ،‬وقد تحتاج هذه العالجات إلى متطلبات إو�جـراءات‬‫)‪ mediated HSCGT‬كعالج لمرض منجي )‪(MNGIE‬‬‫أقــل للمريض ما قبل ز ارعــة نخاع العظم ومــن المتوقع أن‬‫‪173‬‬‫‪ Flt3-L‬بتركيزات منخفضة نسبياً تسببت في نقل الجينات‬‫ع ـنــد تـطـبـيـقـهــا ع ـلــى ال ـف ـئ ـران م ــن ف ـئــة ‪.Tymp-/-Upp1-/-‬‬‫تعطي تصحيح مرضي طويل األمــل‪ ،‬كما هو موضح في‬‫ت ــم اس ـت ـخــدام ال ـفــايــروســات الـبـطـيـئــة )‪ (LV‬ذات التعطيل‬‫الفصل الثاني‪.‬‬‫التلقائي )‪ ،(SIN‬وقــابـلــة للتطبيق عــاجـيـاً‪ ،‬والـتــي تحتوي‬‫في الفصل الثالث‪ ،‬قمنا بتهيئة بيئة خارجية (‪ex vivo‬‬‫‪ )culture conditions‬مثالية لعمليات نقل الجينات من‬‫ـسـرات الـفـسـيــولــوجــي الـمــوجــود‬‫عـلــى محفز كـيـنــاز الــفُـسـفــوغـلـيـ َ‬‫في اإلن ـســان )‪ (hPGK promoter‬من أجــل التعبير عن‬‫خــال الـفــايــروســات البطيئة إلــى الخاليا الجذعية والسلفية‬‫‪ human TYMP cDNA‬األص ـل ــي )‪ (PGK-TP‬أو‬‫المكونة للدم )‪ (HSPCs‬لضمان كفاءة نقل الجينات خالل‬‫المعدل )‪ .(PGK-TPco‬تم استخدام الفايروسات البطيئة‬‫فترة تنبيت قصيرة‪ ،‬وباستخدام أقل عدد ممكن من النواقل‬‫‪ ،LV-SF-TPco‬والتي تحمل محفزقوي )‪ (SF‬لتقييم مدى‬

‫‪Appendix‬‬‫واألن ـمــاط الظاهرية لخاليا الــدمــاغ العصبية (وذم ــة الـمــادة‬‫إضافية قبل القيام بالتطبيق السريري‪ ،‬على أن تشمل تلك‬‫البيضاء والخاليا النجمية)‪ ،‬واالعتالل العضلي في األمعاء‬‫الدراسات تقييم مدى فاعلية العالج الجيني للخاليا الجذعية‬‫(ضمور العضلة المخصوصة) وذلــك من خالل عدد قليل‬‫الدموية )‪ (HSCGT‬في بيئة سريرية أكثر مالئمة تحاكي‬‫نسبياً لنسخ عــامــل )‪ LV-PGK-TP(co‬لـكــل خـلـيــة‪ ،‬من‬‫جسد اإلنـســان المصاب بمرض منجي )‪( (MNGIE‬مثل‬‫دون ح ـصــول أي تـسـمــم جـيـنــي أو تـغـيــر مـظـهــري ظــاهــر‪.‬‬‫ف ـئ ـران مــن فـئــة ‪ Tymp-/-Upp1-/-‬خــاضـعــة لـنـظــام غــذائــي‬‫وب ـس ـبــب وجـ ــود ن ـقــص ف ــي ع ــدد م ــن أه ــم األنــم ــاط الشكلية‬‫يـحـفــز األنـ ـم ــاط ال ـظ ــاه ـري ــة)‪ ،‬وج ــرع ــة م ـعــامــل ال ـفــايــروســات‬‫العصبية أو الـخــاصــة بالجهاز الهضمي لــدى الـفـئـران من‬‫البطيئة )‪ (LV‬والخاليا المزروعة المعدلة جينياً‪ ،‬إضافة إلى‬‫فئة ‪( Tymp-/-Upp1-/-‬مثل االختالل في الوظائف الحركية‬‫إج ـراء تقييمات أكثر عمقاً للتوزيع البيولوجي للفيروسات‪،‬‬‫أو الدنف (متالزمة الـهـزال))‪ ،‬فإنه يتوجب علينا أن نكون‬‫إضــافــة إل ــى د ارسـ ــات أكـثــر عـمـقـاً تـتـطــرق إلمـكــانـيــة حــدوث‬‫حــذريــن عند ترجمة النتائج التي توصلنا إليها عند د ارســة‬‫ت ـس ـم ــم ج ـي ـن ــي (‪ )phenotoxicity‬أو ت ـغ ـي ــر م ـظ ـهــري‬‫الـفـئـران وتطبيقها على المرضى المصابين بمرض منجي‬‫ظاهر )‪.(genotoxicity‬‬‫)‪ .(MNGIE‬بالرغم من ذلك‪ ،‬فإننا ننصح بإجراء دراسات‬‫‪174‬‬

List of terms and abbreviations| |List of terms and abbreviations
AAV
adeno-associated virus
ADA-SCID	 adenosine
deaminase-severe
combined immunodeficiency
ANT-1
adenine nucleotide translocase
type 1BBB	
blood brain barrier
BFU-E
burst forming unit-erythroid
BMbone marrowCD	
cluster of differentiation
cDNA
complimentary DNA
CFU-GM
colony forming
unit- granulocyte monocyte
CGD	
chronic granulomatous disease
CIPO
chronic
intestinal
pseudoobstruction
CIS
common integration sites
CNT
concentrative nucleosidetransporter
CPEO
chronic progressive external
ophthalmoplegia
cPPT
central polypurine tract
CRISPR/Cas clustered regularly interspaced
short palindromic repeats/
CRISPR-associateddCTP
DeoxycytidinetriphosphatedTTPdeoxythymidine triphosphateDNAdeoxyribonucleic aciddNTP
deoxyribonucleoside triphosphates
dThd
thymidinedUrddeoxyuridine
ECGF1
platelet-derived endothelial cell
growth factor
EE-TP
erythrocyte encapsulated TP
ENT
equilibrative
nucleosides transport
ERT
enzyme replacement therapy
FIX	
coagulation factor IX
FACS
fluorescence activatedcell sorting

Flt3-LG-CSF	
GFAP
GFP
γ-RV
GVHD	
Gy
H&E
HIV
HLA
hPGK	
HSCs
HSCT
HSCGT
HSPCs
iPSCs
IRES
LAM-PCR
LHON
LinLV
LVIS
MBP
MDS
MNGIE

MOI
mRNA

FMS-like tyrosine kinase
3-ligandgranulocyte colony
stimulating factor
glial fibrillary acidic protein
green fluorescent protein
gammaretrovirus
graft versus host disease
Gray (irradiation dose unit)
hematoxylin-Eosin
humanimmunodeficiency virus
human leukocyte antigen
humanphosphoglycerate kinasehematopoietic stem cells
hematopoietic stem
cell transplantation
hematopoietic stem cellgene therapy
hematopoietic stem andprogenitor cells
induced pluripotent stem cells
internal ribosome entry site
linear amplification
mediated PCR
Leber’s hereditary
optic neuropathy
lineage negative
lentivirus
lentiviral integration sites
myelin basic protein
mitochondrial DNA
depletion syndrome
mitochondrial
neurogastrointestinal
encephalomyopathy
multiplicity of infection
messenger RNA

A

175

Appendix

mtDNA
NT
OLT
PCR
PEO

mitochondrial DNA
nucleoside transporter
orthotopic liver transplantation
polymerase chain reaction
progressive
external ophthalmoplegia
PLP
proteolipid protein
PTAH
phosphotungstic acid-hematoxylin
qPCRquantitative PCR
RAG -SCID	 recombination activating gene
1 and 2 severe
combined immunodeficiency
RNAribonucleic acidRRE
Rev response element
SCF	
stem cell factor
SCID- X1
X-linked severe
combined immunodeficiency

176

SF	
SIN-LVTALENsTP
TPO
TYMP
TYMPco
UCB	
VCN
VRAC
WAS
WPRE

ZFNs

spleen focus forming virus
self-inactivating LVtranscription activator-like
effector nucleases
thymidine
phosphorylase enzyme
thrombopoietin
thymidine phosphorylase genecodon optimized TYMP geneumbilical cord blood
vector copy number
volume regulated anion channel
Wiskott-Aldrich Syndrome
woodchuck hepatitis
posttranscriptional
regulatory elementzinc-finger nucleases

PhD Portfolio| |PhD Portfolio
Name PhD student: Rana Yadak
Erasmus MC Department: Neurology
Research School: Molecular Medicine (MolMed)
PhD period: Sept. 2011- Dec. 2017
Promoter: Prof.dr. P.A.E. Sillevis Smitt
Supervisors: Dr. N.P. van Til and Dr. I.F.M. de Coo
Year

Workload(ECTS)

General courses
Laboratory animal science
Basic Human Genetics
Basic Introduction Course on SPSS
Research management for PhD students
Research integrity
Biomedical scientific English writing

2012
2013
2013
2014
2015
2016

3
0.5
1
1
0.3
2

Specific courses
5B irradiation protection course

2015

1.4

1- PhD training

Seminars and workshops
Annual MolMed day (2x)2012-2013
NCBI & other open source software
2014
Toxicology of gene-modified hematopoietic cells- Hannover Medical school 2014
Photoshop and Illustrator CS 6 workshop
2014

0.6
1
0.6
0.3

Presentations
NVGCT- Lunteren (3x)NVKN- Alkmaar
ESHG- MilanYMF and Euromit- Tampere
CELL- PID- Ankara
The XXIInd Annual ESGCT congress- The Hague
ASGCT 18th annual meeting - New Orleans
11th EPNS- Vienna
International MNGIE consortium meeting- Innsbruk

2014-2016
2014
2014
2014
2014
2014
2015
2015
2016

3
1
1
1
1
1
1
1
1

Posters
Spierziektecongres- Veldhoven (2x)Euromit- Tampere
ICNC- Amsterdam

2014-2015 0.6
2014
0.3
2016
0.3

National conferences
NVGCT- Lunteren (3x)2014-2016 1.7

International conferences
PERSIST annual EU-project meeting – Leukerbad

2012

A

1.1

177

Appendix
PhD Portfolio (continued)

CELL- PID annual EU-project meeting- Stresa (2x)
ESHG- MilanYMF and Euromit- Tampere
CELL- PID- Ankara
The XXIInd Annual ESGCT congress- The Hague
ASGCT 18th annual meeting - New Orleans 11th
EPNS- Vienna
International MNGIE consortium meeting- Innsbruk
ICNC- Amsterdam

Year

Workload(ECTS)

2012-2013
2014
2014
2014
2014
2015
2015
2016
2016

2.3
1.1
1.7
0.6
1.1
1.1
1.1
0.3
0.3

2013

38

2-Teaching
Supervising a student during her master’s thesis
3-Others
Research visits
Dept. of Immunology, Weizmann Institute of Science (4 months)
Dept. of Pathology, VUMC- Amsterdam (3 months)
Lab of Neuromuscular and Mitochondrial Disorders, Vall d’Hebron
research Institute- Barcelona (3 weeks)

2012
2015,2017
2015

Travel grants
Tebu Bio researchers travel grant to attend ESHG in Milan
Meritorious abstract travel award to attend the ASGCT 18th annual
meeting in New Orleans

2014
2015

Volunteer activity
The XXIInd Annual ESGCT congress-The Hague

178

2014

List of publications| |List of publications
Yadak R, Sillevis Smitt P, van Gisbergen
MW, van Til NP, de Coo IF. Mitochondrial
Neurogastrointestinal Encephalomyopathy
Caused by Thymidine Phosphorylase
Enzyme Deficiency: From Pathogenesis to
Emerging Therapeutic Options. Front Cell
Neurosci. 2017;11:31.
Van Gisbergen MW, Voets AM, Starmans
MH, de Coo IF, Yadak R, Hoffmann RF,
et al. How do changes in the mtDNA andmitochondrial dysfunction influence cancer
and cancer therapy? Challenges, opportunities
and models. Mutat Res Rev Mutat
Res. 2015;764:16-30.
Huston MW, Riegman AR, Yadak R, vanHelsdingen Y, de Boer H, van Til NP, et al.
Pretransplant mobilization with granulocytecolony-stimulating factor improves B-cellreconstitution by lentiviral vector genetherapy in SCID-X1 mice. Hum Gene
Ther. 2014;25(10):905-14.
Khurana S, Melacarne A, Yadak R, Schouteden
S, Notelaers T, Pistoni M, et al. SMAD
signaling regulates CXCL12 expression in
the bone marrow niche, affecting homing andmobilization of hematopoietic progenitors.
Stem Cells. 2014;32(11):3012-22.
Yadak R, Cabrera-Pérez R, Torres-Torronteras
J, Bugiani M, Haeck J, Huston MW, et al.
Preclinical efficacy and safety evaluationof hematopoietic stem cell gene therapy ina mouse model of MNGIE. (Submitted).
Yadak R, Boot M, van Til NP, CazalsHatem D, Finkenstedt A, Bogaerts E, et al.
Transplantation, gene therapy and intestinal
pathology in MNGIE patients and mice.
(Submitted).
Van Til NP *, Yadak R*, Buffa V, Huston
MW, Aerts-Kaya F, van Helsdingen Y, et al.
Development of a highly efficient lentiviralgene transfer protocol for hematopoietic stem
cells. (To be submitted).
*The authors contributed equally
Huston MW, Horsman S, Nowrouzi A, vander Velden G, Varkouhi A, Li Y, Yadak R, et al.
Hematopoietic stem cell lentiviral integration
profiles are stable across multiple species,disease phenotypes and hematopoietic cell
types. (To be submitted).

A

179

Appendix| |Curriculum vitae
Name
Date of birth
Place of birthE-mailPositions
2011-2016
Education
2011-2017

2009-20112004-2008180

Rana Mahmoud AbdulRaheem Yadak
23 March 1986
Nablus, the West Bank, PalestineRana.yadak@gmail.com
PhD position: Dept. of Hematology and Dept. of Neurology, Erasmus
University Medical Center, Rotterdam, the Netherlands
PhD candidate, Biomedical Sciences, Dept. of Hematology and Dept. of
Neurology, Erasmus University Medical Center, Rotterdam, the Netherlands
Thesis: ‘Lentiviral Hematopoietic Stem Cell Gene Therapy for MNGIE’
Promotion date: 19 December 2017
MSc, Biomedical Sciences (Cell and Gene Therapy), Faculty of Medicine andPharmacy, Vrije Universiteit Brussel, Brussels, Belgium,
Thesis: ‘Role of BMP signaling in regulating SDF-1α expression in murine bonemarrow derived stromal cells’
BSc in Medical Laboratory Sciences, Faculty of Science,
An-Najah National University, Nablus, PalestineAcknowledgements| |Acknowledgements
My PhD was a whirlwind of excitement,challenges and achievements. The enormous
help and encouragement from many people
around me made this mission possible.

the MNGIE project. You made me realize,
the hard way, that finding reward in the PhD
process was just as important as publishing
and achieving my career goals.

First of all, I would like to express mysincere gratitude to my promoter Prof.
Peter Sillevis Smitt for guiding me throughthe thesis process, without you, this thesis
would not have come to fruition. Thank you
for accepting me into the Department of
Neurology and becoming my promoter. I am
indebted to your efforts, your kindness andgiving a fair voice when needed. Our meetingsand conversations were always a ray of hope,and your attention to my thesis progress, andtimelines always put me back on track amidst
all the challenges.

I am grateful for my inner doctoral committee,
esteemed Prof. Arnold Vulto, Prof. Jan
Cornelissen, and Prof. Rob Hoeben for taking
the time to read my thesis and provide yourvaluable input. Additionally, I would also liketo thank certain collaborators for their sincere
help in the studies performed in this thesis.
Prof. Marjo van der Knaap, thank you verymuch for providing me with the opportunity
to work with Marianna Bugiani. Max, I verymuch enjoyed working with you on Chapter
5. Thanks to Ian and Chiara, Monique andJoost, George and Elly, Steffi, Ed, Rob, Lucaand Lorena, Dominique and Armin.

To my copromotor Dr. Niek van Til, yourscientific support for the completion of
the studies outlined in this thesis is verymuch appreciated. You taught me the basics
of molecular assays and I enjoyed our brain
storming during experimental design. Things
were tough during my PhD, with you also
having to relocate to Utrecht, and I appreciate
your determination to continue with thisthesis. We learned something new fromeverything we did, both in our successes andfailures, as well as with all the experimentsthat were not pursued; the cytotoxicity, iPSCs,
mobilization protocols, dNTP experiment,
the characterization of the lymphoma...
To Prof. Gerard Wagemaker and mycopromotor Dr. René de Coo, I appreciate
the opportunity you provided to initiate
my PhD, and your attempts to maintain

Thank you Dr. Theo Luider and Peter
Burgers, Jorgen Bierau and Huub, Martijn
for your efforts to set up the HPLC, Dr.
Karoly Szuhai and Danielle for assistancewith the SKY analysis. Prof. Tsvee Lapidot
and team members, I have gained valuable
experience through my visit to your laboratory,
thank you. To Prof. Claudia Waskow, thankyou for your support and valuable advice on
many occasions.

A

Dr. Marianna Bugiani, I very much enjoyedworking with you and I will never forgetyour support throughout my PhD journey.
Marshall, I appreciate your practical guidance
and feedback, and the scientific discussions
we had, as well as your company during myrelocation to the Genetics Department. Your
presence made this transition smooth and181

Appendix

easy. Jeroen, I enjoyed working together
on the isolation of mtDNA. Our brain
storming sessions on MNGIE, in particular
the lymphoma and the AAV gene therapy
projects, which we could not pursue,
were greatly appreciated. Kees, Lidia andJacqueline, you allowed me to perform
experiments in your lab and provided valuableassistance and guidance during the mtDNA
isolation. I enjoyed working in a group withyou all, and Kees, I appreciate our discussions
at the Romit and beyond. Thank you all.
To my former colleagues at the Hematology
department, dear Trui, I learned a lot fromyou, I am forever indebted to your wisdom
and kindness; Leonie and Qiushi, I find it
expressive what Qiushi wrote, ‘it is the mutual
support we have given to each other that havehelped us go thus far in our PhDs’ and personal
lives, and Qiushi, I am proud of us, we madeit through our PhDs. I very much enjoyedhaving you around in the ML-I, -II, bacterialab, the big lab, the animal house. Particularly,
your presence was immensely supportive at
the end of our localization at the Hematology
dept. , as well as our discussions how toface challenges. Your PhD thesis confirmed
to me that it takes more than hard workand dedication to achieve an exceptional
PhD thesis, thanks to your supervisors andteam members who supported your thesis
and projects. To my student Valentina, you
did great with your master’s thesis. I wasproud watching you learning, discussing
your experiments and the results. To Elnaz,
Fatima, Yvette, Guus and Gwen, I appreciate
your assistance, valuable instructions
and feedback. To Merel, thanks for yourcontribution in the beginning of MNGIE

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project, and for saying “no” to me, thus I
am better trained to deal with genotyping
and experimental matters besides solving
the problems independently. To Noemi, Joyce,
Davine, Amiet, Roberto, Adrian, Ferry, andJasper, I enjoyed the occasional talks andsocial activities. Thanks to Eric Bindels andElwin for the technical assistance.
Department of Molecular Genetics, thankyou for hosting me during the last part of
my PhD. Particularly, I would like to thankProf. Roland Kanaar, Prof. Jan Hoeijmakers,
and Dr. Pier Mastroberardino for giving me
the opportunity to finish my experiments
and for valuable advice on many occasions.
Peter, Wilbert, Joris, Renata, Serena, Sander,
Yvette, Yvonne, Silvia, Nicole, Maria, Soraya,
Sabine, Shirley, Meneka, Danny, Jasperina
and the staff of the Molecular Genetics dept.,
I am grateful for the intellectual and technicalsupport whenever I needed it, the very useful
discussions, and the wonderful and upliftingsocial events.
To Diana, Akos, Chiara, Aida, Chang,Ines, Marjolein and Hester, I cannot thankyou enough for everything you have done
for me in the past two years. I am indebted
to you all for our stimulating scientific andpersonal discussions, the lovely gatherings
for international food, multiple celebrations,
the wonderful meetings at Worm and hiking
in Theux and Spa. Thank you, your support
helped me get through my tough journey.
Diana, Akos, Chiara, words cannot express
how thankful and grateful I am for your caring
and support until the last day of my PhD. To
Rajendra, म मेरो यात्रामा तपाईको अनन्त
समर्थनको लागी अनिवार्य छु, हाम्रो यात्रा रAcknowledgementsनेपाल को त्यो यात्रा, र कोरल ड्रा संग सहयोग.
Hugo and Tina, I will always remember our
wonderful hike in the Ardennes and lookforward to having similar trips soon. Martha
and Rui, Amany, Ali, Thabet, Nael, David,
Kfir, Juliet and Ludo, Patrick, and Jorrit,
thank you all very much for your intellectualand emotional support.
To my fellow PhD students, all the best in
finalizing your theses, and just remember
what the great philosopher Nietzsche said,“what does not kill you, makes you stronger.”I also dedicate this work to all the patients
who might eventually benefit directly or
indirectly from the findings outlined inthis thesis.
Palestine, I owe you so much for becoming
who I am now due to the difficult times weexperienced during the second Intifada.
The braveness and stubbornness in goingto school under siege prepared me to face
the challenges I encountered during my PhD.
Dr. Riad Amer and Mr. Nader Asfour, I verymuch appreciate our insightful scientificdiscussions and your support to advance in
my career.

To Nour, we both are at different places yet
always by each other’s side, thanks for being
there for me. Reem, Abud, Dr. Mohammad
and his family, grandmother, aunts anduncles and their families, thank you verymuch for your kindness and prayers.
Last but not least, my beloved parents.
To father, you encouraged me to pursue
higher education, and your support was
immeasurable. You gave me the opportunity
to spread my wings and fly. Thank you,forever. My beloved mother, I cannot
express my gratitude for your kindness andconcern; your voice and the coffee in the earlymornings gave me warmth and the strength tocontinue till the end of the day. To you, I am
forever grateful.
Rana
P.S. There’s more to publishing scientificpapers than good science or advancing
our career as scientists. Behind every wordand between every line lies the curiosity,
dedication, and sacrifice of the PhD students
who helped bring it to fruition. This journey,filled with both tears and laughter, iswhat makes for a rewarding experience inpublishing scientific work.

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Lentiviral Hematopoietic Stem Cell Gene Therapy for MNGIE

The results presented in this PhD thesis demonstrate the efﬁcientand persistent correction of the biochemical phenotype in
affected organs of Tymp-/-Upp1-/- mice, brain neurologicalphenotypes (white matter edema and astrocytes morphology),
and intestinal myopathy (atrophy of the muscularis propria), at
relatively low vector copy numbers and without obvious vector ordisease-related phenotoxicity or genotoxicity. Due to the lack ofsome of the major neurological or gastrointestinal clinical-/-/phenotypes in Tymp Upp1 mice (such as motor dysfunction orcachexia), researchers should be cautious in translating our
ﬁndings from mice to MNGIE patients. Nonetheless, it isrecommended to perform more studies before clinicalapplication, including evaluating HSCGT in more clinicallyrelevant models that mimic human MNGIE disease, the dosing of
LV vector and transplanted gene modiﬁed cells, and a morethorough assessment of vector biodistribution and safety studies
addressing potential phenotoxicity and genotoxicity.

CGACTGCCGAGCTCCGCCCTCCAGGCGGCCCCACCCGCCTGCCGTCCTGGGGCGCCGCCGCCCCGCCGCCGGCAGTGGACCGCTGTGCGCGAACCCTGAACCCTACGGTCCCGACCCGCGGGCGAGGCCGGGTACCTGGGCTGGGATCCGGAGCAAGCGGGCGAGGGCAGCGCCCTAAGCAGGTA
CGGGCGGGGCTCAAGTCGCGAGGCGGGGAAGCGGGAGGCAGACACGGACGAGGGCGACACAGACACGGGACCGAGGGGCGGACACCGGAGAGACACGGGAAAGGGGTCGGGACAGGAGCACGTGGCTCAGACACCGACGCCGGGAGGCCGCAGACCCCGGACGTGTCAGGCA TCCCCGCAGGC
CCGGAGCGATGGCAGCCTTGATGACCCCGGGAACCGGGGCCCCACCCGCGCCTGGTGACTTCTCCGGGGAAGGGAGCCAGGGACTTCCCGACC
CTTCGCCAGAGCCCAAGCAGCTCCCGGAGCTGATCCGCATGAAGCGAGACGGAGGCCGCCTGAGCGAAGCGGACATCAGGGGCTTCGTGGCCG
CTGTGGTGAATGGGAGCGCGCAGGGCGCACAGATCGGTGCGTGGGGAGGGTTGGGCGTTCCTGACCCCGACTGGGAGGTCAGCCCGAGAGACT
TTGGGTCCCTGGGGGTGCGACGGTGCCCCACTACCAGCACCGGCCCCAGGGTGCCCCACCGCTGTGGGCTGCCACCCTCACGCGT ACCCCCAC
ATACCAGGGGCCATGCTGATGGCCATCCGACTTCGGGGCATGGATCTGGAGGAGACCTCGGTGCTGACCCAGGCCCTGGCTCAGTCGGGACAGC
AGCTGGAGTGGCCAGAGGCCTGGCGCCAGCAGCTTGTGGACAAGCATTCCACAGGGGGTGTGGGTGACAAGGTCAGCCTGGTCCTCGCACCTG
CCCTGGCGGCATGTGGCTGCAAGGTTAGAAACCACCTCCTTTCCAGACGGGAGCCTATACCGCACATGCAGCAACCAGTCCATCCACAGGCAGCTCCCAACCTCAAGCCTGGCCCAAAGCCTCCAAGACCCTACCAAGGCTTCTCCCCACCCTGCTCCCCAGCACAGTTCTCCCCACCCCGTTCCCCAGC
ACAGCGCTTGGGGCCCCTCTGGCTCCAGACCAGGCCCCTTGGAGCAGGAAAAAGATCCACTGATGGAATTCAGACCCCTTTCCCCTTGGGTCCCC
AGACAGCTCCCCCAAGGGAGGAGCTGAGGACTTTC CTCCCTCTGCCCCAAGCCTTGTTTCCCCAAGGACAGGTACCAACCTCCTCCCCTACTGAC
ACTTCTCAACCAAGAAAACTTCCTTTCCATTCCCTCACCAGCTGGGCACCCCTATAGCTGCTTAAATACTTTCCAAATCCAGCTGCACTCCTAGCCAG
GGAAGGTGAAGGGATGCACAGAGGTGGGGGAGGGGTACTGTGCAGGGTACTCAGCATCCCTGACCACCAGGTGCCAATGATCAGCGGACGTGGT
CTGGGGCACACAGGAGGCACCTTGGATAAGCTGGAGTCTATTCCTGGATTCAATGTCATCCAGAGCCCAGAGCAGGTACGGGGCGCCACGGATCAGTCATTAATCCAGGTTGATGATGGAGACCCTGGCCAGAATCACTAAAAGATCACTGGTGGATCATTAGGGTCACTAATGAGAACACTGGTCAAGGTTA
CTCATGAGTCACTGGGCCTGGGCCGAAATCATCAGTGGAACTTTGATTAGGATCATAAAATGGGAAGTTGGTCAAAATCACAGATGGCTGGCGGGG
CACGGTGGCTCACACCTGTAGTCCTAGCACTTGGGGAGGCCGAAGAGGGCAGATCCCTTGAACCCAGGAGTTCAAAACCAGCCTGGATAACACGGCAAAACCCCATCTCTACAAAATAGTTCGCTGCGTGTGGTGGTGCACGCATGTGGTTCCAGCTACTCAGGAGGCTGAGGCAGGAGGATCACTTGAGCCTGGGAGGTCTAGGCTGCAGTGAGCCGGGACGATGCCACTGCACTCCAGCCTGGGCAACAGAGTGAGACCCTGTCCCAGCACTCTGGGAGGCAGAGGAGCCCAGTTGGAGATCAGCCTGGGTAATATAGTGAAACTTGATCTCTACAAAAAAAAGAAGAAAAAAAAAAGCCGCGTGTGGTGGTGCGCAC
CTGTAGTCCCAGCTACTGGGAAGCTGAGGTGGGAGGATCACTTAAGCCCAGGAGGCAGAGGTCACAATGAGCCGAAATTGTGCCAACTGCACTCC
AGCCTGGGCAACAGAGGAAGACTCTTCACAGAAAAAAAAAAAAAAAAAAAAAAAGCTGCT AAGTCATTTACCATAAGTCACTGAGAACAGGGGATGTCTGACCAGATGCAAGTGCTGCTGGACCAGGCGGGCTGCTGTATCGTGGGTCAGAGTGAGCAGCTGGTTCCTGCGGACGGAATCCTATATGCAGCCAGAGATGTGACAGCCACCGTGGACAGCCTGCCACTCATCACAGGTGACCTGACTCCATGGCCTGCTTCTGCATGTTCACAGGCTCCTGACCTCCAAACTCAAGTCAAGGGCCTCTCGTTAGGAGTTACCCGTCACCTGACCGTGTGCCCCCCTACCCCCATCACAAGATGCCTGACCACCACCATGTGGGGT
GGCCTGATACTCAACCCACCAGGTGCTGCCACCCCCATAATAAGGGACTTGACCCTCAATGCTCAGGGCCCCTGACCCCAAAGTCGGCATCCCCGAACTCTCCCAAGAAGCTCCAGGTTCTCCATTGTCTCCAACCTCCTCTGCCTCCCCCAAAGCCTCCATTCTCAGTAAGAAACTCGTGGAGGGGCTGTCCGCTCTGGTGGTGGACGTTAAGTTCGGAGGGGCCGCCGTCTTCCCCAACCAGGAGCAGGCCCGGGAGCTGGCAAAGACGCTGGTGAGCGGTGT
GGCCTTTCCCTGGGCAAGCGTCTTGATGCGGGCCCAGCCTACCCTTCACCCCTCCCGTCCCCACTGCCTCCCTCCACTCAGCAGTCCTGCCT AAC
CCCAGTCCCACCCTCTTCTGCCCGAAGTCCCTCCCTCCTTCACGGCTTCCTAACCTGCTGTGACTTTAGAGGTCAAGGCTGGCCCGGCCTGGACCTGGGGAAGCCCTCTGTGGCGTTCCTGCCCCAGACCAAGTACAAGTTCCTCCTGGCCCCATGGCGAGGTGTCGCACTTCACTCGTGTCTCTTCCCC
ACCCCAATCCTTCCCTGACTTCATGCTGGGGGGCTGGCAACCCAGGGTGCAGCAGGGGCTGGAGTTCGACCAAGAACCGGCTGCAGAAGGCCCC
GCCATGGGGGGTCCACGCTGAGCCTCCTCTCCGCAGGTTGGCGTGGGAGCCAGCCTAGGGCTTCGGGTCGCGGCAGCGCTGACCGCCATGGAC
AAGCCCCTGGGTCGCTGCGTGGGCCACGCCCTGGAGGTGGAGGAGGCGCTGCTCTGCATGGACGGCGCAGGCCCGCCAGACTTAAGGGACCTG
GTCACCACGCTCGGTGAGGGGGACGGGGTGTAGGGGAGCGGAGGCGGCGGGGGGTGCTTCCCGCTGGGGCCGCCCCGACCCGGCCGCGCCTAAGACCCGTCCCCGCCCGCAGGGGGCGCCCTGCTCTGGCTCAGCGGACACGCGGGGACTCAGGCCCAGGGCGCTGCCCGGGTGGCCGCGGC
GCTGGACGACGGCTCGGCCCTTGGCCGCTTCGAGCGGATGCTGGCGGCGCAGGGCGTGGATCCCGGTCTGGCCCGAGCCCTGTGCTCGGGAAGTCCCGCAGAACGCCGGCAGCTGCTGCCTCGCGCCCGGGAGCAGGAGGAGCTGCTGGCGCCCGCAGA TGGTGAGCGTCGGGGGAGTCCCCGT
CCTTCCGCCTCCGCCATCCCCTTCCCTTCCCGAGGCCCCGCCCCTTCCCGAGCCCGCGCCTCTCAGCCCCTCTCCCCGCAGGCACCGTGGAGCTGGTCCGGGCGCTGCCGCTGGCGCTGGTGCTGCACGAGCTCGGGGCCGGGCGCAGCCGCGCTGGGGAGCCGCTCCGCCTGGGGGTGGGCGCAGAGCTGCTGGTCGACGTGGGTCAGAGGCTGCGCCGTGGTGAGCGCCGCCCCCGCCCTGCTGGCCCCGCACCCCCGCCCAGCTCCGGCCGCGC
GGCCTCTAACAGCCCCTCGCTCTGCAGGGACCCCCTGGCTCCGCGTGCACCGGGACGGCCCCGCGCTCAGCGGCCCGCAGAGCCGCGCCCTG
CAGGAGGCGCTCGTACTCTCCGACCGCGCGCCATTCGCCGCCCCCTCGCCCTTCGCAGAGCTCGTTCTGCCGCCGCAGCAATAAAGCTCCTTTGC
CGCGAAAGGGCCGGGCGCAGCCGCGCTGGGGAGCCGCTCCGCCTGGGGGTGGGCGCAGAGCTGCTGGTCGACGTGGGTCAGAGGCTGCGCC
GTGGTGAGCGCCGCCCCCGCCCTGCTGGCCCCGCACCCCCGCCCAGCTCCGGCCGCGCGGCCTCTAACAGCCCCTCGCTCTGCAGGGACCCC
CTGGCTCCGCGTGCACCGGGACGGCCCCGCGCTCAGCGGCCCGCAGAGCCGCGCCCTGCAGGAGGCGCTCGTACTCTCCGACCGCGCGCCATTCCCGGGACGGCCCCGCGCTCAGCGGCCCGCAGAGCCGCGCCCTGCAGGAGGCGCTCGT ACTCTCCGACCGCGCGCCATTCTCCCTTCGCAGAGCTCGTTCTGCCGCCGCAGCAATAAAGCTCCTTTGCCGCGAAAGGGCCGGGCGCAGCCGCGCTGGGGTGCGGGCGCAGCCGCGCTGGGGTGCTGCAGGGACCCCCTGGCTCCGCGTGCACCGGGACGGCCCCGCGCTCAGCGGCCCGCAGAGCCGCGCCCTGCAGGAGGCGCTCGT ACTCTCCGACCGCGCGCCATTCCCGGGACGGCCCCGCGCTCAGCGGCCCGCAGAGCCGCGCCCTGCAGGAGGCGCTCGTACTCTCCGACCGCGCGCCATTCT
CCCTTCGCAGAGCTCGTTCTGCCGCCGCAGCAATAAAGCTCCTTTGCCGCGAAAGGGCCGGGCGCAGCCGCGCTGGGGTGCGGGCGCAGCCGC
GCTGGGGTGCTGCAGGGACCCCCTGGCTCCGCGTGCACCGGGACGGCCCCGCGCTCAGCGGCCCGCAGAGCCGCGCCCTGCAGGAGGCGCTCGTACTCTCCGACCGCGCGCCATTCCCGGGACGGCCCCGCGCTCAGCGGCCCGCAGAGCCGCGCCCTGCAGGCGCCGCAGCAA TAAAGCTCCT
TTGCCGCGAAAGGGCCGGGCGCAGCCGCGCTGGGGTGCGGGCGCAGCCGCGCTGGGGTGCTGCAGGGACCCCCTGGCTCCGCGTGCACCGG
GACGGCCCCGCGCTCAGCGGCCCGCAGAGCCGCGCCCTGCAGGAGGCGCTCGT ACTCTCCGACCGCGCGCCATTCCCGGGACGGCCCCGCGC
TCAGCGGCCCGCAGAGCCGCGCCCTGCAGGAGGCGCTCGTACTCTCCGACCGCGCGCCATTCTCCCTTCGCAGAGCTCGTTCTGCCGCCGCAG
CAATAAAGCTCCTTTGCCGCGAAAGGGCCGGGCGCAGCCGCGCTGGGGTGCGGGCGCAGCCGCGCTGGGGTGCTGCAGGGACCCCCTGGCTCCGCGTGCACCGGGACGGCCCCGCGCTCAGCGGCCCGCAGAGCCGCGCCCTGCAGGAGGCGCTCGT ACTCTCCGACCGCGCGCCATTCCCGGG
ACGGCCCCGCGCTCAGCGGCCCGCAGAGCCGCGCCCTGCAGGGCAGAGCCGCGCCCTGCAGGAGGCGCTCGT ACTCTCCGACCGCGCGCCATTCCCGGGACGGCCCCGCGCTCAGCGGCCCGCAGAGCCGCGCCCTGCAGGAGGCGCTCGT ACTCTCCGACCGCGCGCCATTCTCCCTTCGCAGAGCTCGTTCTGCCGCCGCAGCAATAAAGCTCCTTTGCCGCGAAAGGGCCGGGCGCAGCCGCGCTGGGGTGCGGGCGCAGCCGCGCTGGGGTGCTGCAGGGACCCCCTGGCTCCGCGTGCACCGGGACGGCCCCGCGCTCAGCGGCCCGCAGAGCCGCGCCCTGCAGGAGGCGCTCGT ACTCTCCGACCGCGCGCCATTCCCGGGACGGCCCCGCGCTCAGCGGCCCGCAGAGCCGCGCCCTGCAGGCGCGCCCTGCAGGAGGCGCTCGT ACTCTCCGA

Lentiviral Hematopoietic Stem Cell
Gene Therapy for MNGIE

Rana Yadak
2017

Rana Yadak