Faculté des sciences

Département de biologie

Travail personnel de Bloc3 biologie

Can cell and gene therapies’ association treat

bullous diseases?

De Stercke Jonathan
UNamur
2020-2021
Table of contents
1. Introduction: What are bullous diseases............................................................................................. 3
2. Cell therapies ....................................................................................................................................... 7
3. Skin therapies ...................................................................................................................................... 9
3.1. Ex vivo strategies .......................................................................................................................... 9
3.1.1. Viral based ............................................................................................................................. 9
3.1.2. Non-viral based ................................................................................................................... 10
3.2. In vivo strategies......................................................................................................................... 10
3.2.1. Intradermal and intravenous injections .............................................................................. 10
3.2.2. Microneedles ....................................................................................................................... 10
3.2.3. Gene gun-mediated particle bombardment ....................................................................... 11
3.2.4. Nanoparticles ...................................................................................................................... 11
4. Functional therapies .......................................................................................................................... 12
4.1. cDNA replacement therapy ........................................................................................................ 12
4.2. Gene editing therapies ............................................................................................................... 13
4.3. RNA therapies............................................................................................................................. 14
5. Cell and gene therapies’ association ................................................................................................. 15
6. Conclusion ......................................................................................................................................... 17
7. Bibliography....................................................................................................................................... 17

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1. Introduction: What are bullous diseases
“Bullous diseases” is a generic name that comprises various autoimmune and genetic
disorders in which structural components of the epidermal junctions (desmosomes) or
epidermal-dermal junctions (hemidesmosomes) are altered. Autoimmune bullous disorders
include pemphigus, pemphigoid, and IgA-mediated bullous dermatoses (Egami et al., 2020;
Taghipour & Perera, 2013; Venning, 2011) while genetic bullous diseases refer to the various
forms of inherited epidermolysis bullosa (Peking et al., 2018). The structure of a normal skin,
a desmosome and a hemidesmosome is represented below (Figure 1). However, the skin is not
the only organ damaged by bullous diseases. Indeed, other regions such as the oral, the
urogenital and the intestine can be altered (Peking et al., 2018). General features of these
diseases are listed in Table 1.

Figure 1: structure of a normal skin, a desmosome and a hemidesmosome.

(A) The epidermis is composed of 4 layers and the epidermal basement
membrane zone (BMZ) is located between the epidermis and the dermis.
(B) Transmembrane proteins such as desmocollins and desmogleins (Dsg) can
form either homophilic or heterophilic interactions to ensure intercellular
adhesion. Plakoglobin (PG), plakophilin (PKP), and desmoplakin (DP) link the
intermediate filaments to the cytoplasmic domains of desmocollins and
desmogleins. (C) The hemidesmosome is composed of two intracellular
proteins (BP230 and plectin), two transmembrane proteins (BP180 and a6b4
integrin) and two extracellular matrix components (laminin332 and type VII
collagen). The extracellular factors can directly be inhibited by antibodies in
pemphigus and pemphigoid. However, the function of autoantibodies targeting
intracellular factors is unclear (Pecking et al., 2018).

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Table 1 : General features of bullous diseases. Bullous diseases can be divided into two categories: autoimmune or inherited. These categories can further be divided into subcategories
which are detailed in the table. (After Egami et al., 2020; Mariath et al., 2020 ; Parodi et al. ,2020 ;Peking et al., 2018 ; Siegel et al., 2003 ; Taghipour & Perera, 2013; Venning, 2011)

Category Disease name Frequency Localization Altered structural Clinical manifestations

components
Pemphigus: Europe: from 0.5 cases/million/y in Germany to 8 in Epidermal (skin Dsg1 and/or 3 Blisters and dermatitis:
-vulgaris (PV) Greece. USA: incidence is 8-fold higher in the Jewish and/or mucous - mucosal tissues for PV.
-foliaceus (PF) population. South America and North Africa: higher as membranes) - only on the skin for PF.
-paraneoplasic (PNP) well (probably due to a cross-reaction between the sand fly - both in PNP.
salivary antigen LJM11 and Dsg1). Incidence also seems to
be higher among women.

Pemphigoid: No geographical difference in the prevalence. However, the Subepidermal (skin proteins of the Tense blisters, pruritic
Autoimmune

-bullous (BP)
-mucous membrane (MMP)
-epiderpmolysis bulla
acquisita (EBA)
Linear IgA disease
incidence of BP is increasing due to the development of and/or mucous hemidesmosomes erythema and erosions:
diagnostic assays and the aging of the general population. membranes) (BP180 and BP230) -mostly in the limb flexures
and on the abdomen for BP.
-mucous membranes in MMP.
-extensor surface of the
extremities for EBA.
Europe:0.5 cases/million/y Subepidermal (skin) BP180, BP230 and Similar to bullous pemphigoid
Commoner in China, southeast Asia and Africa LAD285
 Epidermolysis bullosa
simplex (EBS)
Junctional epidermolysis
bullosa (JEB)
Inherited (epidermolysis bullosa)
Dystrophic epidermolysis
bullosa (DEB). Can either be
recessive or dominant, the
recessive type being the most
severe phenotype.
Kindler syndrome (KS)
40 cases/million live births Intraepidermal -Intermediate filament Blisters above the dermal-
(skin, mucous genes: Keratin 5 and epidermal junction + muscular
membrane and keratin 14. dystrophy and pyloric atresia
sometimes muscles) -PLEC (plectin gene) when PLEC muted.
2.68 cases/million live births Subepidermal -Laminin 332 protein Skin and mucosal blistering
within the lamina encoded by three genes: upon mechanical stress, hair
lucida of the skin LAMA3, LAMAB3 and loss, nail, dental and enamel
and mucous LAMC2 dystrophy.
membranes -Type XVII collagen
encoded by COL17A1
26.4 cases/million live births Subepidermal- Type VII collagen Skin blistering accompanied
dermal interface encoded by COL7A1. by intense pain, growth
Important for anchoring retardation, delayed puberty,
fibrils  basement anemia, osteoporosis and high
membrane is thus not risk of developing an
connected to the dermis. aggressive squamous cell
carcinoma
<100 cases reported in the world literature attachment of the Kindlin-1 encoded by Skin, oral, urogenital and
actin cytoskeleton KIND1 intestine blistering.
to the extracellular Concerning the skin, this
matrix  causes disease is also associated with
problems wherever photosensitivity, skin atrophy
actin is needed for and skin cancer.
this function (see
clinical
manifestations)
5
 Regarding treatments, systemic corticosteroids combined to immunosuppressive adjuvants Commented [PR1]: Le terme d’adjuvant
immunosuppresseur ne me semble pas correct. N’est-ce pas
are typically applied when dealing with cases of autoimmune origin. Tetracycline, steroid plutôt immunosuppressive agent ?

pulses, plasmapheresis and intravenous immunoglobulins (IVIg) can also be used in some
severe pemphigoid cases but these can have adverse effects (Egami et al., 2020).
Interestingly, new pieces of research suggest that microorganisms may play a role in the
development of autoimmunity. Indeed, the microbiome found near the lesions in bullous
pemphigoid is different from the one found at the same sites in healthy individuals.
A cutaneous microbiome profile that correlates with this disease was also discovered
(Miodovnik et al., 2017) and it seems that mice with a distinct microbiome do not develop
symptoms even though they possess the autoantibodies associated with epidermolysis bullosa
acquisita (Ellebrecht et al., 2016). Targeting dysbiosis could therefore be considered as a
complementary treatment for autoimmune bullous diseases (Zorba et al., 2020). However, this
Commented [JD2R1]: Ecrit comme ça dans l’article.
Coquille dans l’article initial ?
Commented [PR3]: Juste une précision : tu es susceptible
d’être interrogé sur tout ce qui se trouve dans ton travail
écrit (même si ce n’est pas présenté à l’oral). On peut donc
te demander ici d’expliquer comment fonctionne la
tétracycline ? En quoi une administration de stéroides peut-
elle aider dans un cas de pemphigus ? Comment l’injection
d’Ig agit-elle pour soulager une pathologie auto-immune ?
etc
Commented [PR4]: NB : chouette paragraphe !

will not be discussed further since few research has been done on the topic.

No treatment is currently available for inherited epidermolysis bullosa, so clinicians can do

nothing but focus on palliative aspects such as prevention of skin trauma or wound care
(Gonzalez, 2013). Hence, there is a crucial need to develop new strategies to cure these diseases.
Cell and gene therapies seem particularly appealing in this prospect, especially when combined
together (Peking et al., 2018). The first ones, cell therapies, aim to restore a normal phenotype
by using induced pluripotent stem cells (iPSCs) for example while gene therapies try to edit the
genome of the patients in order to completely “delete” the problem (Pecking et al., 2018;
Wenzel et al., 2014). These two approaches and their combination will be further discussed in
the next sections.

Nevertheless, some of these therapies display important limitations like hematopoietic cell
transfer (HCT) for instance. As a matter of fact, not only does HCT requires a human leucocyte Commented [PR5]: Comment un HCT peut traiter une
epidermolysis bullosa ?
antigen-matched donor, it also involves significant risks of morbidity and mortality associated
Commented [JD6R5]: Dans le cadre d’une maladie
with the procedure itself (Webber & Tolar, 2015). Concerning gene therapies, clinical trials d’origine auto-immune : en remplaçant le système
immunitaire du patient pour que le nouveau ne présente pas
performed in the early 2000s with the aim of curing X-linked severe combined d’auto-anticorps vis-à-vis des protéines des desmosomes,
etc.
immunodeficiency (X-SCID) showed adverse events such as the development of leukemia in
five out of the 20 patients treated in France and the UK back then (Zhang et al., 2020). Commented [PR7]: ? que veux-tu dire ?
Commented [JD8R7]: A l’époque
 Since then, newer treatments are being investigated notably with the development of several
CRISPR/Cas9 molecules (Peking et al., 2018) or induced pluripotent stem cells (iPSC)
(Wenzel et al., 2014) for example. Advantages and drawbacks of these new perspectives will
thus be assessed in this work to try answering whether it is possible to cure inherited bullous
diseases using a combination of cell and gene therapies.

2. Cell therapies
Several cell therapies have been tried as off today. For instance, wounds in recessive
dystrophic epidermolysis bullosa (RDEB) may be treated with HCT, which has been
successfully performed in some RDEB patients (Wagner et al., 2010). However, this procedure
has significant risks of mortality so palliative care (bandaging, itch and pain control, etc.) is the
only option widely available to RDEB patients nowadays (Webber & Tolar, 2015; Wenzel et
al., 2014).
To overcome these problems, novel cell therapies such as fibroblast cell therapy or
recombinant type VII collagen have been developed. Concerning the fibroblast cell therapy,
promising results have been shown in mice where COL7A1-overexpressing human fibroblast
have been injected intravenously. Indeed, these fibroblasts can migrate to skin wounds where
they generate anchoring fibrils by delivering wild-type type VII collagen (C7) (Woodley et al.,
2007). Other pieces of research displayed that intradermal injections of allogeneic fibroblasts
and mesenchymal stromal cells (MSCs) have the potential to address the cutaneous
manifestations in RDEB patients by expressing C7. However, these injections don’t have any
effect on the underlying systemic manifestations associated with the various forms of EB.
Furthermore, the cell populations used do not contain stem cells so the benefits are only
transient (Webber & Tolar, 2015). A protein therapy using recombinant type VII collagen,
injected intravenously, have also been tried in mice where it restored type VII collagen
expression. (Woodley et al., 2013). However, this has not been tried on humans yet so it will
not be discussed further in this paper.

Placenta-based therapies are also under development for the treatment of EB. Even if they
make use of stem cells just like HCT, these therapies seem particularly interesting. Indeed, they
drastically reduce the risks of infection transmission from donor to patient, need a less stringent
human leukocyte antigen (HLA)-match and display lower risks of graft-versus-host disease
(GVHD). Moreover, MSCs derived from umbilical cord blood (UBC) are easier to isolate, have
a faster doubling time in vitro and are more available nowadays thanks to the increasing

7
prevalence of cord blood banking (Nevala-Plagemann et al., 2015). Surprisingly, even a partial
restoration of C7 may provide clinical benefits as shown in a murine RDEB model where
autologous MSCs were transplanted into the skin. Indeed, only 15% or wild-type C7 levels
were able to prevent the separation along the dermal-epidermal junction (DEJ) which typically
occurs in RDEB patients (Alexeev et al., 2013). Other pieces of research even claim that UCB
and placenta-derived MSCs may have a greater immunosuppressive potential than other sources
of MSCs (Nevala-Plagemann et al., 2015). Consequently, using them to treat autoimmune
bullous diseases might be of interest. Amniotic membrane grafting has also been performed for
chronic wounds on RDEB patients and showed promising results as well as four out of eight
patients displayed a significant clinical response and one was even completely healed (Lo et al.,
2010).
Finally, cord blood platelet (CBPG) trials have also been performed and showed promising
results for the treatment of fresh cutaneous wounds in patients suffering from EB (Figure 2).
However, the effect on chronic wounds was almost non-existing. Therefore, CBPG would be
more suited for treating newborns suffering from EB early on (Gelmetti et al., 2018). Moreover,
treating oral lesions with CBPG is still a challenge even though combining it with
photobiomodulation therapy (PBMT) can reduce intraoral discomfort by diminishing the
lesion’s size and pain (Sindici et al., 2018).

Figure 2: Lesions before and after 5 CBPG applications in a DEB patient (Gelmetti et al., 2018).

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3. Skin therapies
Even though cell therapies do seem interesting, these cannot cure the diseases in the long run.
Thus, researchers have also been trying to edit the mutation using gene therapies in order to
find a cure. In this prospect, they have been trying to target human epidermal stem cells these
haven the potential to regenerate an entire epidermis. Several approaches, either ex vivo or in
vivo, have since been developed to this end (Figure 3).

Figure 3: examples of functional therapies for the treatment of bullous diseases. In ex vivo therapies, the patient's
cells are biopsied and then corrected so that they can be re-injected into the patient. On the other hand, in vivo
therapies seek to correct the problem by directly modifying the patient's defective cells (after Peking et al., 2018).

3.1. Ex vivo strategies

3.1.1. Viral based
Vectors derived from the murine leukemia virus (MLV) were the first used in an attempt to
treat cure genetic diseases such as adenosine deaminase deficiency which causes severe
combined immunodeficiency (ADA-SCID). Unfortunately, these viruses are known for their
capacity to integrate into actively transcribed DNA which led some patients to develop cancer
due to their therapy because of insertional mutagenesis (Pecking et al., 2018; Zhang et al., 2020).
Using self-inactivating (SIN) vectors can help decreasing this risk because they lack enhancer
or promoter elements in their long-terminal repeats (LTR). Moreover, SIN vectors have already
been used to restore the COL7A1 gene associated with RDEB (Titeux et al.,2010). Of course,
risks of insertional mutagenesis still exist but detecting cancers prematurely on the skin is way
easier than in the bone marrow for example, which makes viral vectors a lot safer in the context
of bullous diseases than in ADA-SCID therapies (Pecking et al., 2018).

9
3.1.2. Non-viral based
Toxicity and immunogenicity can be a major hurdle when using viral vectors. Thus, non-
viral transfer techniques, such as plasmids which are also less expensive, have been developed.
However, transfection efficiency is lower compared to viral-based strategies but selecting cell
clones after treatment can help bypassing this problem. Another advantage of using non-viral
plasmids is that they can be designed to carry enhancer elements, tissue-specific promoters and
other regulatory elements required for transgene expression in the transfected cells (Pecking et
al., 2018).

3.2. In vivo strategies

Although ex vivo approaches display several advantages such as the possibility to assess the
safety before transplantation or to target stem cells, these also have certain drawbacks. Indeed,
ex vivo strategies require to graft cells onto the patient which is obviously more invasive as
anesthesia is needed. Chronic wounds in EB also are a problem since they cause depletion of
stem cells over time. Consequently, developing in vivo strategies do seem appropriate as well,
if not more than ex vivo ones, for the treatment of EB (Pecking et al., 2018).

3.2.1. Intradermal and intravenous injections

Using injections as gene therapies has not been done on humans yet but promising results
have been made in DEB xenograft mouse models where antisense oligonucleotides (ASO) were
injected subcutaneously to induce COL7A1 exon 73,80 and 105 skipping. Indeed, as these
exons are often mutated in DEB, skipping them could drastically improve the phenotype of
patients suffering from this disease (Bremer et al., 2016). However, this method displays two
main drawbacks: (1) in-frame exons are the only ones that can be skipped which limits the
number of patients that could be treated this way and (2) the method is time consuming and
very expensive at the moment (Bornert et al., 2017).

3.2.2. Microneedles
At first, solid microneedles were used to make the skin more permeable to drugs. Since then,
other microneedles have been developed such as microneedles directly coated with drugs,
polymer microneedles containing the drug that can dissolve within the skin and hollow
microneedles which are used to infuse the drug into the skin (Figure 4). Overall, these newer
generations of needles improve the procedure’s safety and the patient’s well being compared to
classic hypodermic needles which are more commonly used (Kim et al., 2012).

10
Figure 4: Methods of drug delivery using microneedles (MN) for skin therapies. Nowadays, four types of MN
exist: solid MN, coated MN, dissolving MN and hollow MN. Solid MN are used to make the skin more permeable
to drugs which are applied topically after. Coated MN are directly coated with a drug. Dissolving MN are made
out of polymers that can dissolve into the skin in order to release the drug. Finally, Hollow MN can be used to
infuse a drug into the skin (Kim et al., 2012).

3.2.3. Gene gun-mediated particle bombardment

Historically, gene guns approaches were only used in plants to facilitate transformation of
totipotent tissues such as pollen or embryos. Technically speaking, gold particles coated with
DNA are pressurized with gas in these guns. This enables the particles to penetrate tissues at
high speed without damaging them in order to transform the target cell’s genomes (Pecking et
al., 2018). Concerning the treatment of EB using this method, a COL7A1 repair molecule has
already been efficiently delivered into the skin of a wild-type mouse model. Moreover, no
blister formation was observed due to this technique which potentially makes it appropriate as
a treatment for the extreme fragile skin of EB patients (Pecking et al., 2016).

3.2.4. Nanoparticles
In healthy skins, the stratum corneum is a natural barrier against particle penetration.
However, this is less the case in diseased skins such as the ones found in EB patients (Prow et
al., 2011). With this in mind, researchers fused nanoparticle polymers to non-viral vectors
expressing full-length COL7A1 complementary DNA and tried to restore the skin of mice
suffering from RDEB. In this study, expression of type VII collagen was visible at the BMZ 5
days post application and still detectable after 10 weeks (Cutlar et al., 2016). Unfortunately,
adverse events are difficult to predict because many types of nanoparticles are available
(organic, inorganic or polymers) and their safety has not been sufficiently investigated yet (Nitta
& Numata, 2013; Pecking et al., 2018).

11
4. Functional therapies
Many functional therapies are currently being investigated to treat EB. Among these, three
strategies appear to be promising: cDNA replacement, gene editing and RNA targeting which
are summarized in Figure 5 (Pecking et al., 2018).

Figure 5: Functional therapies that could be used to treat EB. Three common strategies to restore gene function
are listed here:(A) cDNA replacement, (B) Gene editing and (C) RNA therapy (after Pecking et al.,2018).

4.1. cDNA replacement therapy

The first gene replacement therapy aiming to cure EB was performed in 2006 in a patient
suffering from JEB because of a LAMB3 gene mutation. In this study, keratinocytes were
isolated from the patient’s palm and then transduced using a retroviral vector expressing full-
length LAMB3 cDNA. Strikingly, complete epidermal regeneration and sustained synthesis of
LAMB3 proteins were observed even 7 years after transduction (Pecking et al., 2018). Another
study demonstrated that cDNA could also be used to treat RDEB patients. Unfortunately, the
corrected grafts didn’t last very long in this trial. This suggests that treating RDEB with this
approach could be done but requires better wound bed preparation or the removal of resident
uncorrected cells for example (Nyström & Bruckner-Tuderman, 2016).

12
4.2. Gene editing therapies
Using designer nucleases such as transcription activator like effector nuclease (TALEN) or
clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9
nuclease (CRISPR/Cas9) seems particularly interesting in order to cure EB because (1) EB is a
monogenic disease and (2) designer nucleases do not share the risks associated with viral
vectors. Their mechanism of action is summarized in Figure 6 (Pecking et al., 2018).

Figure 6: Mechanism of action of (A) CRISPR/Cas9 and (B) TALEN. In both methods, gene editing starts by
targeted double-strand breaks (DSB) at the gene locus which needs to be deleted or corrected. CRISPR/Cas9
editing requires (1) the presence of a protospacer adjacent motif (PAM) sequence near the cleavage site and (2)
guide RNA (gRNA) binding on the target site which activates the Cas9 nuclease. The DSB is then corrected by
non-homologous end-joining (NHEJ). On the other half, the DSB is preceded by two TALEN dimerization.
Homology-directed repair (HDR) is then performed to correct the DSB. (After Pecking et al., 2018).

Both mechanisms can correct specific sequences and thus, subsequently, protein expression.
However out-of-frame products can also be generated because of random errors during the
processes, especially with NHEJ (Santiago et al., 2008). Consequently, HDR is preferable for
precise genome editing. Moreover, TALEN can be used to target any sequence with the only
requirement being that a thymine needs to be located at the beginning of the binding site.
Still, CRISPR/Cas9 is more commonly used because generating these molecules is less
complex and time consuming than designing TALENs (Pecking et al., 2018). Anyhow, off-
target effects do exist and prevent possible clinical applications of designer nucleases.
Nevertheless, researchers are trying to bypass this problem by selecting the corrected cell clones
ex vivo or by improving the nucleases specificity using mutant versions of the Cas9 protein, for
instance (Pecking et al., 2018).

13
4.3. RNA therapies

Interfering with post-transcriptional processes such as mRNA splicing could also be a good
option for the treatment of EB. Among these approaches, antisense oligonucleotides (ASO)-
mediated exon skipping and spliceosome-mediated RNA trans-splicing (SMaRT) seem
particularly appealing (Pecking et al., 2018). ASO have already been described with
intradermal and intravenous injections, the rest of this section will not focus on these. Basically,
RNA trans-splicing molecules (RTMs) need to be manufactured in order to perform SMaRT.
This mechanism can then hijack the natural splicing machinery of the cell to replace mutated
regions in the pre-mRNA by its wild-type version. SMaRT displays several advantages
compared to other ASO. Indeed, a broad range of patients could potentially be treated with
SMaRT because of two main aspects: (1) a wide variety of RTMs can be designer and (2) they
can be applied both to recessive and dominant forms of EB (Pecking et al., 2018; Wally et al.,
2012).
Apart from ASO and SMaRT, which are splice-modulating therapies, premature termination
codon (PTC) readthrough compounds could also be used to bypass mutations at RNA level.
Indeed, approximately 30% of DEBs are caused by mutations that converts an amino acid into
a PTC. Aminoglycosides seem particularly interesting in this prospect since de novo synthesis
of type VII collagen have already been observed in patients suffering from DEB (Pecking et al.,
2018). Concerning the mechanism of action, aminoglycosides bind to ribosomal RNA which
leads to the introduction of an alternative amino acid during translation (Cogan et al., 2014; Lee
et al., 2012).
Finally, small interfering RNAs (siRNAs) are also applicable but solely for the dominant
forms of inherited EB. As a matter of fact, siRNAs can target specifically the mutant allele in
order to knock it down. Unfortunately, this approach requires patient-tailored molecules which
is still too expensive nowadays for large scale applications (Pecking et al., 2018).

14
5. Cell and gene therapies’ association

In the past decade, some pieces of research aimed to combine cell and gene therapies in order
to get the best out of it. Among these strategies, genetically corrected iPSCs and genetically
modified autologous epidermal grafts seem particularly promising for treating RDEB and JEB
respectively (Nanba, 2019; Wenzel et al., 2014).
Concerning iPSCs, the biggest advantage compared to other cell therapies is that autologous
cells can directly be generated from iPSCs, therefore avoiding the risks of rejection. Moreover,
iPSCs can be cultured and thus potentially provide an unlimited supply of progenitor cells
needed for local and systemic treatment of RDEB. Because these cells can differienciate into
all three germ layers and be corrected by genome editing tools such as the one discussed
previously (TALEN, CRISPR, etc.), iPSCs also permits patient-specific approaches (Wenzel et
al., 2014). Figure 7 displays one possible approach to generate iPSCs in mice that could
potentially be used for humans.

Figure 7: Generation of iPSCs from control and COL7A1 mutant RDEB mice. (A) In this study, dermal fibroblasts
from wild-type (WT) and COL7A1 mutant (Mut) mice were used to generate iPSCs. Firstly, a lentiviral
reprogramming was performed in order to convert fibroblasts into iPSCs. Then, a recombinase (Flpo) was used to
correct mutant iPSCs in order to express WT type VII collagen in all colonies. Individual clones were then traced
with a luciferase reporter and iPSCs were cultured in vitro in order to differienciate into fibroblasts again. A
classical intradermal cell therapy was then performed using these fibroblasts. (B) Time course studies revealed
that the luciferase activity was still detectable up to 16 weeks after the final injection which further highlights the
potential long term benefits this therapy could have on RDEB patients (after Wenzel et al., 2014).

15
 After showing the long-term in vivo survival of corrected fibroblasts, Wenzel et al. then
tested whether type VII collagen was indeed restored correctly in COL7A1 mutant RDEB mice.
The results are displayed in Figure 8.

Figure 8: Restoration of type VII collagen deposition in RDEB mutant mice using genetically repaired iPSC-
derived fibroblasts. (A) Type VII collagen mRNA and (B) secreted protein levels in whole-skin samples show that
type VII collagen expression is indeed restored in RDEB mice that received the treatment. However, (C) and (D)
show the limitations of this approach in the long run since luciferase and type VII collagen mRNAs are no longer
detected after 18 weeks (after Wenzel et al., 2014).

As explained before, genetically corrected iPSCs represent an interesting innovating

approach for the treatment of RDEB. However, phenotype correction does not stay forever
because type VII collagen mRNAs are no longer detected after 18 weeks. In order to bypass
this problem, readministering cells at 18 weeks or increasing the survival of fibroblasts could
be an option. Since these have never been tested, future studies will have to address whether it
is safe or not (Wenzel et al., 2014).

Regarding the treatment of JEB, genetically modified autologous epidermal grafts represent
another innovative approach that could help cure patients. Indeed, skin samples can be extracted
from the patient in order to genetically correct them as discussed previously (retroviral vectors,
genome editing, etc.) so that skin cells re-express LAMB3. These cells can then be multiplied
in vitro and transplanted onto surgically prepared wound beds (Nanba, 2019). Recent studies
showed that engrafted corrected keratinocytes maintained their populations for at least 6,5 years
and that the method could restore up to 80% of the total body surface area (De Rosa et al., 2014;
Hirsch et al., 2017).

16
6. Conclusion

7. Bibliography
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marrow-derived mesenchymal stem cells and the skin: Ccl27-Ccr10 axis as a basis for targeting to
cutaneous tissues. Cytotherapy, 15(2), 171-184.

Benati, D., Miselli, F., Cocchiarella, F., Patrizi, C., Carretero, M., Baldassarri, S., ... & Larcher, F. (2018).
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Chinen, J., & Puck, J. M. (2004). Successes and risks of gene therapy in primary
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Cogan, J., Weinstein, J., Wang, X., Hou, Y., Martin, S., South, A. P., ... & Chen, M. (2014).
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Cutlar, L., Zhou, D., Hu, X., Duarte, B., Greiser, U., Larcher, F., & Wang, W. (2016). A non‐viral gene
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