Artcile summary/review
Need a article summary in NEW York Times style article format of 1000 words or less. Needs to written so that anybody who read it could uderstand.
Gene Therapy for Autosomal Dominant Disorders of Keratin
Alfred S. Lewin, Peter M. Glazer,w and Leonard M. Milstonez
Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, Florida, USA; Departments of wTherapeutic Radiology
and zDermatology, Yale University School of Medicine, New Haven, Connecticut, USA
Dominant mutations that interfere with the assembly of keratin filaments cause painful and disfiguring epidermal
diseases like pachyonychia congenita and epidermolysis bullosa simplex. Genetic therapies for such diseases
must either suppress the production of the toxic proteins or correct the genetic defect in the chromosome. Because
epidermal skin cells may be genetically modified in tissue culture or in situ, gene correction is a legitimate goal for
keratin diseases. In addition, recent innovations, such as RNA interference in animals, make an RNA knockdown
approach plausible in the near future. Although agents of RNA reduction (small interfering RNA, ribozymes, triplex
oligonucleotides, or antisense DNA) can be delivered as nucleotides, the impermeability of the skin to large
charged molecules presents a serious impediment. Using viral vectors to deliver genes for selective inhibitors of
gene expression presents an attractive alternative for long-term treatment of genetic disease in the skin.
Key words: keratin/ribozyme/siRNA/triplex-forming oligonucleotides/zinc-finger proteins
J Investig Dermatol Symp Proc 10:47–61, 2005
Gene-based therapies depend on several critical elements.
First, one must have a disease gene. ‘‘Having a disease
gene’’ implies having an understanding of the diseasecausing
mutations and how they contribute to pathology.
Second, one must have a therapeutic gene, which may be a
wild-type (normal) version of the disease gene or may be
another gene that suppresses the disease phenotype, for
example, one whose product prevents apoptosis. Third,
gene therapy requires an efficient delivery system. This delivery
system may be a virus or a formulated nucleic acid,
but should permit specific expression of the therapeutic
gene according to cell type. Finally, one should have an
animal model that recapitulates both the genetic and the
phenotypic characteristics of the disease and permits testing
of potential treatments. Animal models include knockout
or transgenic mice and spontaneous mutations leading to a
disease of domestic animals that is analogous to the human
disorder. In this review, we will use these criteria to evaluate
the prospects of gene therapy for dominant keratin diseases,
emphasizing innovations such as transcriptional silencing
and RNA interference as the most promising genetic
therapeutics in the near term.
Keratin Mutations—Disease Genes
Mutations in keratin genes cause oral, ocular, epidermal,
and hair-related diseases (Corden and McLean, 1996;
Fuchs and Cleveland, 1998; Coulombe and Omary, 2002;
Kirfel et al, 2003; Porter and Lane, 2003). The study of these
keratin diseases has contributed to our understanding of
the function of intermediate filaments in epithelial cells, but
the large number of these genes, more than 50, indicates
that the study of keratin biology will continue to be fruitful for
many years to come (Hesse et al, 2004). Pairs of type I and
type II keratins are co-expressed in a developmentally regulated,
tissue-specific manner and form heterodimers that
are assembled into filaments. These intermediate filaments
are the main stress-bearing cytoskeletal components within
the affected epithelial cells. Consequently, the pathology
associated with keratin mutations is determined by the expression
pattern of the defective proteins. For example,
type I keratin K16a and type II keratin K6a are co-expressed
in the palmoplantar epidermis and the orogenital squamous
epithelium. They are also induced by wounding. Missense
mutations in either of these proteins may result in
pachyonychia congenita (PC), which is characterized by
hypertrophic nail dystrophy and palmoplantar keratoderma,
although not by defects in wound healing. Most of the disease-
causing mutations in keratin are missense mutations,
and most affect conserved sequences at the termini of the
central rod domains of the molecules (Coulombe and
Omary, 2002). These regions are probably important for
correct assembly of the keratin filaments.
Nearly all of the human keratin disorders are associated
with dominant mutations. Such an inheritance pattern is
typically caused either by haploinsufficiency or by toxic
gain-of-function mutations. Haploinsufficiency implies that
a single normal allele does not provide a sufficient amount
of protein to support cell viability or function. A common
example is polycystic kidney disease associated with
premature termination mutations in the PKD1 gene that affect
about one in 1000 people of European decent. Most
Abbreviations: AAV, adeno-associated virus; dsRNA, doublestranded
RNA; LTR, long terminal repeat; ODN, oligodeoxynucleotides;
PC, pachyonychia congenita; RISC, RNA inducible
silencing complex; shRNA, short hairpin RNA; siRNA, small interfering
RNA; TFO, triplex-forming oligonucleotides; ZFN, zinc-finger
nuclease
Copyright r 2005 by The Society for Investigative Dermatology, Inc.
47
patients have loss-of-function mutations in one allele of
PKD1. Toxic gain-of-function mutations are caused by either
missense or chain termination mutations that result in
the accumulation of abnormal proteins. In some cases,
these proteins lead to death of the affected cells. Such is
the case with rhodopsin mutations leading to autosomal
dominant retinitis pigmentosa that lead to the apoptotic
death of rod photoreceptor cells. Dominant mutations may
also disrupt the assembly of multimeric protein complexes.
Examples include cardiac myosin heavy chain mutations
leading to familial hypertrophic cardiomyopathy and collagen
I defects leading to osteogenesis imperfecta. Keratin
diseases such as epidermolysis bullosa simplex and PC are
dominant for this reason: missense mutations interfere with
the assembly of multimeric keratin filaments.
Gene Therapy for Dominant Diseases—Why
Supplementation Will Not Suffice
The genetic approach to treating such a dominant disorder
is more complex than that for a simple recessive disease
caused by the lack of a particular protein. In the case of
toxic mutations, it does not suffice to replace a missing
function by delivering a ‘‘healthy’’ gene. Rather, the therapy
must block the production of the defective gene product.
The first approach for blocking protein production employed
antisense RNA and DNA, and this was followed
shortly thereafter by the use of ribozymes. Such approaches
to therapy fall into two categories—nucleotide therapies
and gene therapies. The first class includes antisense
oligonucleotides, triplex-forming oligonucleotides (TFO),
and short double-stranded interfering RNA. The second
class comprises genes that encode nucleotide inhibitors
such as small interfering RNA (siRNA) and ribozymes and
also genes for proteins that may repress the transcription of
specific genes or enhance directed gene repair. Although
this approach to gene therapy has lagged behind that for
recessive diseases, several new technologies and delivery
systems are making gene silencing for keratin disorders
seem plausible.
Epidermal Stem Cells
The accessibility of the skin and the characterization of its
stem cells will facilitate the development of gene-based
therapies for epidermal keratin diseases (Watt, 2000; Potten
and Booth, 2002). Nevertheless, the compartmentalization
of the epidermis and its continuous self-renewal pose obstacles
for long-term therapy that can be overcome only by
genetic transduction of epidermal stem cells. The bulge region
of adult hair follicles contain multipotent progenitor
cells that can reconstitute a wounded epidermis and
respond to morphogenic stimuli by forming hair follicles,
sebaceous glands, and epidermis (Oshima et al, 2001;
Panteleyev et al, 2001). But using retroviral tagging,
Ghazizadeh and Taichman demonstrated that the contribution
of hair follicles to the epidermis was restricted to the rim
of epidermis surrounding the follicle and that the interfollicular
epidermis was independent of follicular stem cells.
Their work suggests that there may be multiple stem cell
lineages within the epidermis and that some of these have
restricted cell-fate in the absence of injury (Ghazizadeh and
Taichman, 2001). The epidermis of the mouse is comprised
of functionally distinct proliferative units, consisting of a
stem cell in the basal layers of the dermis and its dividing
cell progeny, which give rise to a column of differentiated
keratinocytes (Gambardella and Barrandon, 2003) (Fig 1). In
the mouse, this ‘‘column’’ can be two to four cells. Longterm
gene therapy for keratin diseases must target these
stem cells and yet deliver genes that are appropriately regulated
until the progeny of these cells differentiate (Ghazizadeh
et al, 2002). In contrast, oligonucleotide-based gene
silencing should affect the differentiated keratinocytes
themselves. Such treatments will require periodic re-application.
These issues have been discussed in earlier excellent
reviews (Khavari and Krueger, 1997; Somani et al, 1999;
Spirito et al, 2001).
Gene therapies for dominant disease can alter gene expression
at two levels: at the level of the gene (chromatin) or
at the level of RNA (transcript) (Fig 2). Approaches at the
gene level encompass gene correction, gene ablation and
gene silencing. At the RNA level, ribozymes, siRNA, or antisense
DNA can be used to cleave mutant transcripts or to
block their translation into protein. Interposing at the level of
gene should be superior to intervening at the level of the
transcript, as the expression of the disease-causing allele
can be blocked completely and perhaps permanently.
Gene Correction—The Gold Standard of
Gene Therapy
Correcting the mutation that leads to disease is the ultimate
objective of gene therapy (Richardson et al, 2002). This approach
is superior to gene supplementation (i.e., adding an
Figure 1
Columnar organization of the epidermis
into discrete units with individual stem cells.
Based on Alonso and Fuchs (2003).
48 LEWIN ET AL JID SYMPOSIUM PROCEEDINGS
extra wild-type cDNA via virus-mediated transduction) because
the repaired gene will reside at the normal chromosomal
locus and be regulated by its own promoter and
regulatory elements. Despite this consensus among gene
therapists, this ‘‘gold standard’’ has been difficult to
achieve. Several approaches have been attempted in tissue
culture and even in animals. These include using RNA–
DNA oligonucleotides, single-stranded DNA oligonucleotides,
homologous replacement with small DNA fragments,
TFO, and repair with the single-stranded adeno-associated
virus (AAV). As the mechanism by which mutations are corrected
(recombination, primed replication, mismatch repair,
etc.) has not been elucidated, this approach has been referred
to as ‘‘gene targeting’’ or ‘‘gene correction.’’ Without
genetic selection, the frequencies of gene repair of endogenous
chromosomal genes by these methods is generally
low (106–105 per cell), especially in vivo. But the ability to
induce double-stranded breaks in the target gene using
site-specific endonucleases or TFO has increased the probability
of successful gene repair.
Oligonucleotide-Based Gene Targeting
RNA–DNA oligonucleotides (also called RDO or chimeroplasts)
are designed to induce single nucleotide changes in
homologous DNA sequences in mammalian cells. The original
design of RDO contained two strands of about 25 nucleotides
identical in sequence to the target except at the site
of the host mutation; one strand containing both ribo- and
deoxyribonucleotides and a complementary strand containing
only DNA (Cole-Strauss et al, 1999). This method
has been particularly successful in correcting mutations in
episomes for which selection was possible, but has also
been used for the modification of chromosomal genes. For
example, RDO were used to convert the factor IX gene in
hepatocytes and for the repair of the sickle-cell hemoglobin
in lymphoblasts. Subsequently, it was determined that short
DNA oligonucleotides of 40–50 nucleotides have the same
capacity for inducing DNA repair as do RDO. Oligodeoxynucleotides
(ODN) have been used to correct an integrated
copy of the lacZ gene in mouse embryonic stem cells
(Pierce et al, 2003) and to modify the c-kit and the tyrosinase
gene in single melanocytes from albino mice (Alexeev et al,
2002). In this case, the co-conversion of the tyrosinase gene
gave a visual marker by which cells undergoing repair events
could be screened. Transcription apparently facilitates the
formation of intermediates in ODN-directed repair, and the
non-transcribed strand is more frequently the target of correction,
as antisense ODN appear more potent than those
with sense strand polarity (Igoucheva et al, 2003).
Although high levels of gene repair were reported in the
liver, the reproducibility of gene repair by RDO has been
questioned because some researchers have failed to achieve
a significant level of gene alteration. As pointed out by Yoon
et al (2002), the supposition that gene correction using
oligonucleotides would be quick and easy was naive. Large
amounts of oligonucleotides are employed, and quality control
is essential. Highly sensitive assays are required, and
these must be properly controlled. Different cell types have
different repair competence, probably based on the activities
of recombination and repair enzymes. Given the low frequency
of oligonucleotide-directed gene alteration, a selection
procedure is essential to make the gene-repair technology
practical. This implies an ex vivo approach will be required in
which stem cells are manipulated, and those with repaired
genes are expanded and re-introduced into the tissue.
Homologous Replacement with DNA
Fragments
As an alternative to oligonucleotide-stimulated gene repair,
small fragment homologous replacement uses double-
Figure 2
Gene therapy for dominant diseases should either correct a mutant gene or suppress the synthesis of its product. Such therapies target
either chromatin in the nucleus (DNA) or ribonucleoprotein in the cytoplasm (RNA). At the DNA stage, the ideal approach is gene correction, which
may be mediated by oligonucleotides, DNA fragments or adeno-associated virus (AAV). Alternatively, genes may be knocked out using the same
approaches or silenced using triplex-forming oligonucleotides or zinc-finger transcription factors. In the cytoplasm, antisense RNA (with RNase H) or
ribozymes or siRNA can be used to digest transcripts and lead to their degradation. Alternatively, antisense DNA or triplex-forming oligonucleotides
can interfere with the translation of the mRNA.
10 : 1 OCTOBER 2005 GENE THERAPY OF KERATIN DISORDERS 49
stranded DNA fragments to restore a normal sequence to
mutated genes. In this method, 400–800 bp segments of
DNA are introduced into cells, typically using electorporation
(Goncz et al, 2002; Gruenert et al, 2003). This DNA
contains the wild-type sequence and is intended to replace
or repair the mutant gene, although the exact mechanism is
not known. In a model system, a 4 bp insertion in the Zeocin
resistance gene was corrected, concomitantly restoring antibiotic
resistance and an XmaI restriction site (Colosimo
et al, 2001). Homologous replacement has been used to
correct the dystrophin gene in the mdx mouse model of
muscular dystrophy (Kapsa et al, 2001), the b-globin gene in
human hemopoietic progenitor cells (Goncz et al, 2002) and
the cystic fibrosis transmembrane conductance regulator in
human airway epithelial cells from cystic fibrosis patients
(Sangiuolo et al, 2002). As with ODN-stimulated gene repair,
homologous replacement requires introduction of large
amounts of DNA, and efficiency is low (1 correction event
per 105–107 cells).
Targeted Correction with AAV
Because of the low rate of gene correction using DNA fragments
and oligonucleotides, Russell and Hirata (1998) and
Russell et al (2002) have used AAV to stimulate repair. AAV
has demonstrated considerable advantages for conventional
gene therapy (Flotte, 2004), but because it efficiently
delivers single-stranded DNA to the nucleus, it can also
serve as a donor for normal genetic information. For the
type I collagen (COL1A1) gene, up to 1% of normal human
fibroblasts were corrected using gene targeting by AAV (Hirata
et al, 2002). AAV-mediated gene targeting can introduce
1 or 2 bp substitutions and even insertions up to 1.5
kb (Hirata and Russell, 2000; Inoue et al, 2001). Thus, in
cells the frequency of correction using AAV is much higher
(10,000-fold) than that typically obtained by other gene
targeting methods. It should be pointed out that these results
were obtained in tissue culture and rely on phenotypic
selection for correction events. In the absence of selection,
much lower correction frequencies (0.006%) were observed
in primary mouse fibroblasts and in the tibialis muscles of
mice (Liu et al, 2004). Nevertheless, Chamberlain et al (2004)
used AAV vectors and selection for G418 resistance to
specifically disrupt a mutant allele of the COL1A1 gene in
mesenchamal stem cells from patients with osteogenesis
imperfecta. As heterozygous mice that carry disrupted keratin
genes showed no obvious structural and functional defects
of the skin, it is likely that such a targeted disruption
approach could be used to block the expression of mutant
keratin 6a or keratin 16 in stem cells from patients with PC-1.
Two technologies have been used to increase repair by
oligonucleotides, TFO and site-specific endonucleases.
Both triplex-forming ODN and site-specific endonucleases
generate double-strand breaks near the mutation site and
therefore increase recombination repair. TFO can also be
used to block the transcription of DNA.
TFO
Triplex DNA was first recognized in 1957, but only during the
past 15 y has this structure been exploited to interfere with
gene function. TFO are single-stranded oligonucleotides
that form triple-stranded DNA by binding in the major
groove of polypurine: polypyrimidine runs in the DNA duplex.
The TFO binds to the purine strand of duplex DNA with
high affinity and according to a recognized binding ‘‘code.’’
Mismatches in this code generally destabilize the triple helix,
but because triplex formation does tolerate some sequence
variation, much effort has been directed toward
developing nucleotide analogues that would extend the
third-strand-binding code so that triple helix formation
would not be restricted to homopurine or homopyrimidine
runs (reviewed in Gowers and Fox, 1999).
TFO have been used in a variety of ways as sequencespecific,
DNA targeting reagents (reviewed in Knauert and
Glazer, 2001). TFO have been linked to reactive moieties to
produce site-specific cleavage of DNA and to block enzymes
that cleave or modify DNA. For example, by interfering
with RNA polymerase binding and mRNA elongation,
they can inhibit transcription.
Gene Targeting Using TFO
Heritable alterations in DNA by TFO were first demonstrated
using a TFO linked to a psoralen molecule (Takasugi et al,
1991). Site-specific mutations were identified initially in triplexes
formed ex vivo and subsequently in triplexes formed
in episomal and then chromosomal DNA targets. These
psoralen-TFO experiments established the concept that
DNA-binding molecules can be used to direct site-specific,
heritable genome modification. Equally important, they
showed that cell and nuclear membranes and the packaging
of DNA into chromatin presented no absolute barriers to
gene targeting with TFO. In the course of those studies, it
became apparent that TFO alone, even in the absence of
linkage to a psoralen, could induce site-specific mutations.
Mechanistic studies demonstrated that TFO binding to DNA
stimulates DNA repair synthesis, and that intact DNA repair
pathways are required for the production of TFO-induced
mutations (Knauert and Glazer, 2001).
The potential utility of TFO as gene therapy reagents was
greatly expanded by the recent discovery that TFO stimulate
recombination (reviewed in Seidman and Glazer, 2003).
TFO stimulate intrastrand, chromosomal DNA recombination
at high frequency (1%) following intranuclear injection
but at considerable lower frequency when delivered to the
culture medium (Luo et al, 2000). TFO increase the frequency
of donor oligonucleotide-directed, sequence-specific
base changes in DNA in close proximity to the triplex
(Chan et al, 1999). The concomitant delivery of a homologous
‘‘donor’’ oligonucleotide and an adjacent TFO raises
the frequency of donor-mediated, sequence-specific
changes at least 5-fold. In studies that have been done to
date, higher frequencies of targeting were observed if the
donor was covalently tethered to the TFO than when the
oligonucleotides were unlinked, and double-stranded donors
gave higher frequencies of correction than singlestranded
donors.
It is important to acknowledge several potential obstacles
to general use of TFO as gene targeting reagents, and
to recognize that progress is being made in addressing
50 LEWIN ET AL JID SYMPOSIUM PROCEEDINGS
each of those issues (Seidman and Glazer, 2003). Even with
synthetic oligonucleotide mimetics, the binding code limits
the sequences that can be targeted, and intracellular pH
and ion concentration conspire to reduce binding coefficients
below those achieved in ideal solution. Physical,
chemical, and metabolic barriers limit delivery and biological
effectiveness of oligonucleotides, as indicated by the
intranuclear injection experiments cited above. Finally, all
gene targeting methods are prone to a low frequency of
error, which may be introduced during recombination-mediated
repair or by non-homologous end joining. Errors introduced
by TFO appear to be limited to the vicinity of the
TFO-binding site.
The existence of a triplex site at the beginning of the K6a
gene makes it a reasonable model in which to explore and
expand the usefulness of TFO-mediated gene targeting.
The ideal treatment for PC would be one that selectively
repairs or inactivates the mutant K6a allele, leaves the normal
K6a allele intact, incurs no risk of genomic alteration in
sequences other than the targeted sequence, and can be
accomplished with reasonably high efficiency, so that a
lasting effect can be achieved after a limited number of
treatments. But it is possible that gene targeting could inactivate
the wild-type allele only, which for some genes
would make the disease phenotype worse.
Several considerations recommend a TFO-mediated
gene inactivation strategy rather than a correction strategy.
First, complete inactivation of K6a would probably be curative,
as there appear to be highly related genes with redundant
function; animals lacking a single K6 gene have no
clinical phenotype. Second, the inactivation strategy should
work in individuals harboring mutations anywhere in this
gene. Third, as discussed elsewhere in these Symposium
Proceedings, there are reasons to believe that an improvement
in phenotype might occur if even fewer than 100% of
keratinocytes had their mutant allele inactivated; moreover,
the K6a gene might be one of those genes for which cells
having one wild-type and one inactivated allele might have a
selective growth advantage over cells that retain a mutant
allele. Fourth, biologically active oligonucleotides can be
given to humans with few serious risks or side effects. Finally,
a correction strategy was dismissed for the following
reasons. There is no polypurine run in the vicinity of the
known mutations in the human K6a gene. Furthermore, as
all gene targeting strategies have some inherent error rate, if
an error, rather than a correction, were introduced at one of
those critical, helix-coding regions of a keratin gene, an
exacerbation in (dys)function might occur; errors in the noncoding
50 end of the coding sequence are much less likely
to have deleterious effects.
The fortuitous polypyrimidine/polypurine run at the
beginning of exon 1 in the K6a gene suggests a gene inactivation
strategy that utilizes a TFO to stimulate oligonucleotide-
mediated introduction of a stop codon early in the
coding sequence of K6a (see Fig 3).
Gene Targeting Stimulated by Double-
Strand Breaks—Zinc-Finger Nucleases
(ZFN)
On the supposition that gene repair is mediated by homologous
recombination, several groups have used site-specific
endonucleases to cleave double-stranded DNA near
the site of mutation and to induce correction by doublestrand
break repair (Resnick and Martin, 1976). The rare
cutting endonuclease from yeast, I-SceI, stimulates homologous
recombination at the HPRT locus (Brenneman et al,
1996), and delivering this enzyme increases the level of
plasmid-based gene correction events to 1% (Donoho et al,
1998). This approach also promotes gene correction by
AAV. Miller et al (2003) engineered a site for the I-SceI endonucleases
within the lacZ gene and integrated this modified
marker into the genome of human cell lines using a
retrovirus. Delivering the gene for Sce-I with a second retrovirus
increased the frequency of AAV-mediated gene repair
in this system by 60–100-fold. Similar results were
obtained by Porteus et al (2003) who repaired a chromosomally
integrated but defective allele of GFP.
Figure 3
The introduction of a premature stop
codon in K6a using a triplex-forming
oligonucleotide (TFO) and donor oligonucleotide.
The TFO and donor oligos
can be uncoupled or tethered together by
a flexible linker.
10 : 1 OCTOBER 2005 GENE THERAPY OF KERATIN DISORDERS 51
To make this double-strand break repair relevant to gene
therapy, two groups have recently shown that site-specific
endonucleases can be engineered using zinc-finger DNAbinding
domain (Bibikova et al, 2003; Porteus and Baltimore,
2003). The Cys2–His2 zinc-finger is comprised of 30
amino acids that fold into a bba structure stabilized by the
coordination of a zinc cation. It is one of the most common
protein motifs encoded in the human genome. This structure
recognizes a 3-nucleotide sequence in the major
groove of double-stranded DNA and makes contacts mainly
in one strand. Several research groups have used the
principles of rational design or selection on phage displays
to generate zinc-finger domains with novel specificities
(Dreier et al, 2000, 2001; Liu et al, 2002). In this way, zincfinger
domains have been identified with high affinity for all
of the 50GNN30 and many of the 50ANN30 trinucleotides.
This adaptability of zinc-finger domains has been employed
to generate sequence-specific nucleases for gene
targeting. For example, Bibikova et al (2003) designed a pair
of zinc-finger domains linked to a non-specific nuclease
(Fok I) that must dimerize in order to cleave the DNA (Smith
et al, 2000) (Fig 4). Each zinc-finger domain recognizes a 9
bp DNA sequence, so that the total recognition sequence is
an unambiguous 18 bp. Coupling the zinc-finger domains to
the nuclease using a spacer sequence provides a high
specificity ZFN. They used an 8 kb mutant donor fragment
homologous to the Drosophila yellow (y) gene and containing
the cut site for the ZFN. When expression of the donor
fragment and both components of the dimeric nuclease
were co-expressed in embryos, a high frequency (2%) of
yellow progeny was recovered, and most of these were the
result of homologous recombination. Porteus and Baltimore
(2003) have also used a ZFN to stimulate targeted repair of
the GFP gene in a human cell line. In a promising recent
development, Urnov et al (2005) used ZFN to achieve correction
of a SCID mutation in the IL2Rg gene in 18% of
treated human cells in the absence of phenotypic selection.
The stimulation of recombination by double-strand
breaks is a property of all nucleated cells, so that the use
of designed ZFN to improve the frequency of gene repair
should be widely applicable. As noted above, gene repair
must occur in stem cells in order to be sustained in tissue. It
is unlikely that single base-pair substitutions can be discriminated
by ZFN, so that cells in which the mutant chromosome
has been repaired must be selected and
transplanted into the patient. To identify and expand these
cells, some selectable marker, for example an antibiotic resistance
gene, may be included in the flanking wild-type
donor DNA. This gene would be co-transferred with the
wild-type keratin sequence at some frequency, a phenomenon
called co-conversion (Orr-Weaver et al, 1988). As including
a selectable marker decreases the sequence
identity between the mutant chromosome and wild-type
donor DNA, the frequency of gene repair can be expected
to drop significantly.
Zinc-Finger Proteins for Transcriptional
Silencing
Zinc-finger proteins can be used to regulate gene expression
in a variety of ways (reviewed in Jamieson et al, 2003).
In a common application, activation or repression domains
are combined with site-specific DNA-binding domains to
produce novel transcriptional regulators. Typically, the p65
domain from the transcription factor NF-kB or the VP16
domain from herpes simplex virus is used for activating
transcription. Zinc-finger DNA-binding domains can also be
used to inhibit transcription by adding the KRAB domain,
which creates a zinc-finger transcriptional repressor that
mimics the naturally occurring KRAB proteins (Thiesen et al,
1991). Designing a zinc-finger transcription factor involves
identifying an accessible region of the promoter. In practical
terms, this means identifying nuclease sensitive and hypersensitive
sites in the upstream (50) portion of the gene.
These DNA sequences can be used as bait to identify zincfinger-
binding domains in a phage display library of such
motifs (Rebar and Pabo, 1994). Three or more zinc-finger
domains can then be assembled to recognize a 9 bp site.
Dissociation constants should be in the low nanomolar
range. As with the ZFN, dimerization domains can be used
to join two zinc-finger proteins, and to increase the recognition
specificity. Creating these proteins requires several
areas of expertise and is not off-the-shelf technology.
Zinc-finger proteins have been used in this way to stimulate
transcription of the erythropoietin (Epo) and vascular
endothelial growth factor genes (Snowden et al, 2003). Zincfinger
repressors have reduced transcription of CHK2 gene
(Tan et al, 2003) (which encodes a protein phosphatase required
for cell cycle progression) and the peroxisome
proliferator activated receptor-g. As inhibitors of gene
expression, zinc-finger repressors have potential in blocking
synthesis of toxic gain-of-function proteins, such as
mutant keratins. They are at least as potent as another
fashionable technology, siRNA. The catch is that transcriptional
repressors will limit production of both mutant and
normal keratin alleles, as the mutations leading to disease
do not affect the promoter region. Blocking total production
of a specific keratin may be acceptable, if another filament
protein can replace it in the affected cell lineage. As
Figure 4
Targeted gene repair using a zinc-finger endonuclease. Although
this scheme shows the chromosomal sequence as the nuclease target,
the donor sequence may also be cut.
52 LEWIN ET AL JID SYMPOSIUM PROCEEDINGS
indicated, mice deficient for K6a and K6b are phenotypically
normal because of the presence of a third K6 gene.
Alternatively, a synthetic transcription factor can be employed
to block production of both alleles of an affected
gene, for example, and a wild-type cDNA can be supplemented
using a different regulatory sequence that permits
cell-specific expression. Such a strategy might be pursued
most effectively ex vivo followed by re-introduction of genetically
modified cells.
Blocking Expression at the RNA Level—
Antisense Technologies
Although gene correction and transcriptional silencing have
theoretical appeal, blocking gene expression by preventing
translation may be as effective. More importantly, technology
for knocking down mRNA transcripts—antisense
oligonucleotides, ribozymes, and RNA interference—are
closer to application in the near future. Each of these methodologies
has been exhaustively reviewed, so that in this
summary we will only indicate the strengths and weaknesses
of each approach as it applies to PC and other inherited
keratin diseases.
Antisense ODN
The use of antisense ODN, now 25 y old, is the oldest of
these technologies (reviewed in Crooke, 2004). Antisense
technology exploits oligonucleotide analogs (typically
around 20 nucleotides) that bind complementary RNA sequences
through conventional base pairing, resulting in the
destruction or inactivation of the target RNA. Although some
antisense oligonucleotides block gene expression by preventing
protein synthesis, it is clear that the most active
ODN lead to the RNase H-mediated degradation of target
mRNA. RNase H degrades the RNA strand of an RNA–DNA
duplex. It has been identified in all eukaryotic cells and is
encoded by some viruses. DNA sequences as short as four
nucleotides activate RNase H.
The design of antisense ODN is important. Antisense
ODN are typically added as modified oligonucleotides to
increase their stability in cells and tissues, but oligonucleotides
with sugar modifications resembling RNA, such as 20-
fluoro or 20-methoxy, do not serve as substrates for RNase
H. In addition, some backbone modifications influence the
ability of oligonucleotides to activate RNase H. Methylphosphonates
are not substrates of the enzyme whereas
phosphorothioates are excellent substrates. But some chimeric
oligonucleotides that bind to RNA do activate RNase
H. For example, oligonucleotides comprising 20-O-methoxyethyl
phosphonate sequences flanking a five-nucleotide
stretch of deoxyoligonucleotides bind to their target RNA
and activate RNase H. Such molecules were shown to
have essentially identical potency and specificity as siRNA
in a head to head comparison using four different human
mRNA and 80 different hybridization positions (Vickers et al,
2003).
Phosphorothioate-modified ODN have been used successfully
to block the expression of a variety of viral and
cellular genes in tissue culture and in animals (Dean et al,
1996; Dean and Bennett, 2003). Antisense oligonucleotides
have also been employed to modify the splicing patterns of
human b-globin pre-mRNA as a potential therapy for thalassemia
(Suwanmanee et al, 2002). ODN are attractive as
therapeutics because several antisense compounds are already
approved and on the market and especially because
their formulation and pharmacodynamics have been studied
extensively (Crooke, 2004). Of particular relevance to PC
is that antisense ODN can be applied in a skin cream and
permeate the epidermis and dermis in animals and in humans.
Epidermal hyperproliferation in psoriasis has been
reversed by the use of antisense ODN specific for the insulin-
like growth factor I mRNA (Wraight et al, 2000). Alicaforsen
(Isis Pharmaceuticals, Carlsbad, CA), an inhibitor of
intercellular adhesion molecule 1, accumulated throughout
the dermis and epidermis after topical administration and
has shown promise in a Phase II clinical study undertaken
by Isis Pharmaceuticals. It should be noted that the efficiency
of topical delivery is very low, however.
Ribozymes
Ribozymes are catalysts comprised of RNA (Doudna and
Cech, 2002). Eight naturally occurring ribozymes have been
discovered, and each is involved in cleaving and/or forming
a phosphodiester bond in RNA or DNA, sometimes as part
of the replication cycle of an RNA virus or satellite RNA,
sometimes as a step in autocatalytic splicing and sometimes
as part of a retrotransposition event. In vitro selection
has resulted in additional RNA and DNA enzymes capable
of catalyzing novel reactions, including the formation of
peptide bonds and the aminoacylation of tRNA. The small
hammerhead and hairpin ribozymes derived from tobacco
ringspot virus satellite have found the greatest number of
applications for gene therapy. These ribozymes can catalyze
hydrolysis of second RNA molecules at a variety of
cleavage sites: Hammerhead will cleave after the dinucleotide
UX, as long as X is not a guanosine. Hairpin ribozymes
cleave in the sequence BNGUC, where B is not adenosine,
and N is any nucleotide. Such sequences are found in any
mRNA. Once a target RNA is cleaved by a ribozyme, it is
rapidly degraded by cellular nucleases. Like antisense
ODN, ribozymes have been applied for inhibiting the expression
of dominant disease genes, the replication of RNA
and retroviruses and the expression of oncogenes (Lewin
and Hauswirth, 2001).
Ribozymes have a theoretical advantage and two
practical disadvantages with respect to antisense oligodeoxyribonucleotides.
The advantage is that they exhibit
enzymatic turnover and therefore can block the accumulation
of an mRNA in sub-stoichiometric amounts. In the one
study in which ribozymes and antisense inhibitors were directly
compared, ribozymes reduced the target RNA between
2-fold and 10-fold more efficiently than antisense
RNA depending on the target site (Hormes et al, 1997). The
disadvantages are that ribozyme RNA is unstable and relatively
expensive to synthesize, whereas DNA is stable and
cheap. The use of modified nucleotides does stabilize
ribozymes significantly, but at a cost of catalytic activity.
10 : 1 OCTOBER 2005 GENE THERAPY OF KERATIN DISORDERS 53
Synthesis of large amounts of RNA with necessary protective
groups on their 20 carbons and on the 30 termini, is, at
least by current standards costly (dollars per residue), compared
with synthesis of DNA, either modified or straight
(cents per residue). Consequently, ribozymes may have
limited application as nucleotide therapeutics relative to
antisense ODN and siRNA. Instead, ribozymes will be more
effective as gene therapeutics. Following delivery with a recombinant
virus or other delivery vector, ribozymes can be
produced continually from within the target cell.
Because they are easy to design, hammerhead ribozymes
are the most commonly used for gene therapy and
hypothesis testing (Fritz et al, 2002). Basically one chooses
an RNA sequence of 12 or 13 nucleotides with the target
sequence (UX) in the middle and designs an antisense RNA
complementary to that sequence, leaving the ‘‘X’’ unpaired
and resulting in two hybridizing arms of the ribozyme (Fig 5).
The 22 nucleotide catalytic domain is attached to antisense
sequence so that both hybridizing arms have six nucleotides
or the 50 arm has five nucleotides and the 30 arm of the
ribozyme has six. Using a longer hybridizing sequence increases
the affinity of the ribozyme for its target but reduces
the enzymatic turnover, because it retards release of the
cleaved product. Such ribozymes function as stoichiometric
inhibitors. Similar considerations apply to the hairpin ribozyme,
but the target site is more highly conserved.
Although design of hammerheads and hairpins is
straightforward, finding highly active ones is not. Hammerhead
target sites preceded by the trinucleotides GUC, AUC,
CUC, and UUC and followed by the dinucleotides UU or UA
tend to be the best substrates (Shimayama et al, 1995;
Clouet-d’Orval and Uhlenbeck, 1997). Hybridizing arms that
form base pairs with nucleotides in the ribozymes catalytic
domain (and not with the target) lead to inactive ribozymes.
In such short RNA, stable alternative structures can be predicted
reliably using folding programs such as Mfold 3.0
(http://www.bioinfo.rpi.edu/applications/mfold/old/rna/). Although
most non-viral mRNA are not highly structured,
some ribozyme target sites may be buried within a stable
secondary structure element such as a hairpin loop. Computer
prediction of the structure of long RNA (4100 nucleotides)
are unreliable, but stable local folding can be
identified. In the end, experimental verification of the accessibility
of the target site must be obtained using RNA
structure assays (e.g., sensitivity to nuclease or singlestrand-
specific modifying reagents) or by testing the ribozymes
on full-length mRNA in vitro or in cells. As catalytic
turnover defines a ribozyme’s utility, multiple turnover
(substrate excess) kinetic assays using oligonucleotide
substrates are employed to identify the ribozymes with
the highest activity, particularly at low magnesium concentrations.
This is not a technology that lends itself to a kit.
Ribozymes do have an important advantage compared
with antisense ODN and siRNA—they are more highly sequence
specific. A single nucleotide substitution within four
residues of a hammerhead cleavage site will block activity
completely. In antisense oligonucleotides that are 20–25
nucleotides in length, single mismatches will lower affinity
marginally but not prevent inhibition of a wild-type target.
Similarly, 19–23 nucleotide siRNA are frequently insensitive
to as many as three mismatches with a non-cognate target
RNA. Consequently, should a ribozyme target site be located
near the site of a disease mutation, allele-specific
ribozymes could be generated to selectively eliminate the
product of the disease gene. A ribozyme specific for the
F174S mutation of K6a is shown in Fig 5.
The fact that ribozymes can be allele specific does not
necessarily make them ideal for treating a dominant genetic
disease unless there is a predominant allele causing most of
that disease and unless there is a cleavage site associated
with that mutation. This is not the case for the keratin diseases.
Consequently, it may be desirable to develop a ribozyme
that cleaves both mutant and wild-type keratin
mRNA. Such a ribozyme should cleave a site not affected
by the majority of disease mutations (i.e., away from boundaries
of rod domain). Because the genetic code is degenerate,
it is possible to produce a ribozyme-resistant cDNA
for the keratin target and use this as part of an ‘‘ablate and
switch’’ approach to reduce the level of toxic protein and
replace it with normal protein (Fig 6). This method was suggested
for rhodopsin mutations by Millington-Ward et al
(1997) and has been tested in hepatocytes to replace a
dominant-negative form of a-1-antitrypsin (Zern et al, 1999).
It should also be applicable to RNA replacement using si-
RNA. As noted above, as mice deficient in keratin 6 exhibit
no clinical phenotype, the ‘‘replacement’’ part of this
strategy with the ribozyme- or siRNA-resistant gene may
be unnecessary.
RNA Interference
The inhibition of gene expression (RNAi) using doublestranded
RNA (dsRNA) has stimulated a revolution in
molecular genetics, first in plants and in the nematode Caenorhabditis
elegans and more recently in mammalian cells.
This amazing scientific story has been amply recounted.
Several recent papers (Dorsett and Tuschl, 2004; Mittal,
2004; Dillon et al, 2005; Huppi et al, 2005; Shankar et al,
2005) review the use of siRNA in mammalian cells. A further
paper reviews the relationship between micro-RNA (miRNA)
Figure 5
A hammerhead ribozyme specific for the F174S allele of K6a,
which leads to pachyonychia congenita. The target sequence is
shown in red, and the wild-type sequence is above it in black.
54 LEWIN ET AL JID SYMPOSIUM PROCEEDINGS
and the RNA interference apparatus (Murchison and Hannon,
2004).
Long dsRNA is toxic to mammalian cells through the
protein kinase R and the 20-50 oligoadenylate pathways (together
referred to as the interferon response). These pathways
lead to an arrest of protein synthesis and to apoptotic
cell death and are a component of our defense mechanism
to prevent the spread of viruses. For this reason, long
dsRNA fragments are not used to induce specific RNA interference
in mammalian cells. When a short dsRNA (o30
bp) is artificially introduced into mammalian cells, it may
have one of three effects. If it forms a perfect (or nearly
perfect) match with the sequence of an endogenous mRNA
on one of its two strands, it will lead to degradation of that
mRNA using a ribonucleoprotein complex called RISC (RNA
inducible silencing complex). Degradation of the cellular
RNA is what is meant by RNA interference. If the dsRNA
contains short segments of sequence disparity from the
target RNA, it may block translation of that transcript, particularly
if one strand of the dsRNA can bind in the 30 untranslated
region of the mRNA. An additional impact of the
dsRNA may be the induction of transcriptional silencing
through chromatin modification. This phenomenon has
been well studied in plants and in fission yeast but is
thought to occur in human cells as well.
dsRNA also arises endogenously in mammalian cells
through the processing of micro-RNA. Micro-RNA are produced
as RNA stem loops and are processed by an endonuclease
called Dicer into 21–23 nucleotide duplexes with
50 phosphate groups and 30 single-stranded overhangs of
two or three nucleotides. These dsRNA associate with RISC
that unwinds the duplex RNA in an ATP-dependent reaction.
There are over 200 miRNA encoded in the human genome,
and, based on analogy with nematodes, these are thought
to be important regulators of development and differentiation.
Micro-RNA block the translation of their target mRNA.
Most bind in the 30 UTR of these targets (in the few cases in
which those genes have been identified) and form discontinuous
helices.
RNA interference, using exogenous siRNA, exploits the
endogenous miRNA machinery. There is no fundamental
difference between the two pathways: miRNA mutated to
form a perfect match with the target RNA lead to cleavage
by RISC, and introduced dsRNA that contain mismatches
can block translation. Furthermore, Drosophila and other
animals that maintain a specific RNAi pathway for defense
have multiple Dicer enzymes. Mammals have only one—
that used by miRNA and usurped by geneticists introducing
siRNA.
siRNA and Short Hairpin RNA (shRNA)
Short interfering RNA can be introduced to cells and animals
either as RNA or as DNA clones that code for the
dsRNA. Usually, when clones are used, they produce
shRNA that is processed by Dicer to form siRNA in the
cell. For tissue culture work and temporary dosing to tissue,
Figure 6
An RNA replacement strategy using
ribozymes. A ribozyme is designed to
cleave both mutant and normal RNA,
leading to the turnover of these transcripts
by cellular nucleases. Silent mutations
are introduced into a cDNA
making it ribozyme-resistant (‘‘hardened
WT’’). The genes for the ribozyme and the
hardened wild-type are introduced together.
This same strategy can apply to
siRNA induced RNA interference.
10 : 1 OCTOBER 2005 GENE THERAPY OF KERATIN DISORDERS 55
direct delivery of siRNA has distinct advantages: Short RNA
molecules are easily synthesized and are commercially
available in pure form. dsRNA is more stable than singlestranded
RNA and can be rendered even more stable by
chemical modifications in the sense strand of the duplex.
Such modifications have the added advantage of increasing
the incorporation of the antisense strand of the duplex into
RISC, therefore increasing cleavage of the intended mRNA.
Using RNA also permits accurate dosing of the cells, as an
exact amount of nucleic acid is added. The disadvantage of
siRNA treatment is that large amounts of RNA are required
(typically 10–100 nM final concentration) and the effect is
temporary (three to four cell generations). Using a high dose
of siRNA may saturate Dicer and other components of the
miRNA apparatus and lead to non-specific effects, by preventing
the activity endogenous miRNA. Identifying highly
active siRNA should reduce this side effect.
Plasmids and viral vectors can be used to deliver shRNA,
which are usually produced under the control of RNA polymerase
III (pol III) promoters. These promoters generate
large amounts of short RNA, but most is sequestered in the
nucleus away from the cytoplasmic RISC. Consequently,
some investigators have switched to pol II promoter systems
for the expression of shRNA (Xia et al, 2002) or have
disguised their siRNA hairpins as micro-RNA to facilitate
transport to the cytoplasm (Boden et al, 2004). Delivery with
viral vectors has the advantages that hard-to-transfect primary
cells can often be infected and that expression of the
RNA hairpin may remain stable over many cell generations,
particularly if an integrating virus is employed. The massive
effort focused on tissue-specific targeting of viral vectors
should facilitate both systemic and site-specific delivery of
shRNA. The use of plasmid or viral vectors also facilitates
the generation of shRNA libraries, which are of use in functional
genomics. In addition, delivery as DNA provides a
relatively inexpensive way to screen a series of siRNA for
their potency as inhibitors, compared with RNA duplexes,
which currently cost over $250 each. As even modified duplex
RNA is degraded in tissue, vector-based therapies will
provide a continuous supply of siRNA, although the dosage
may be difficult to estimate. Some hairpin RNA have been
shown to stimulate the 2050 oligoadenylate pathway, even if
they are kept short, and this represents a serious disadvantage
to the delivery of siRNA as shRNA. In general, such
‘‘off target’’ effects are a pitfall of all RNAi methods and
must be controlled for in experiments.
Designing siRNA
Most artificial siRNA contain 19 nucleotides of duplex RNA
plus two unpaired nucleotides at the 30 end (Fig 7). Adding
50 phosphate is optional, as the duplex will be phosphorylated
once inside the cell. RNA hairpins may be a bit longer,
and are generally connected by a loop of six or more nucleotides
based on the structure of a naturally occurring mi-
RNA. (Most investigators feel that the loop sequence is of
limited importance.) There are no absolute rules for selecting
target sites for siRNA, but there are some guidelines.
These are based on the lessons derived from hundreds of
siRNA molecules tested in cells (Reynolds et al, 2004; Ui-Tei
et al, 2004) and from the discovery that siRNA are loaded
onto RISC asymmetrically (Khvorova et al, 2003). In general,
any part of the mRNA can serve as a target, but regions of
extremely high (450%) or low (o30%) guanosine plus
cytosine (GþC) content should be avoided. Sequences
containing ‘‘G quartets’’ or stretches of four or more As are
also poor substrates. Although global RNA folding programs
are poor predictors of mRNA structure, and thus are
useless in siRNA design, tight hairpins that form within the
target RNA will be mimicked in the strands of the siRNA and
may block RNA interference.
Other guidelines for siRNA design reflect the bias of
RISC: There should be high thermal stability (high GþC) at
the 50 end of the sense strand (the strand with the same
polarity as the mRNA) and low stability (high AþU) at the 30
end of the sense strand in order to promote incorporation of
the antisense strand into RISC (which proceeds in the 50 to
30 direction). Low thermal stability in the middle of the
duplex seems to promote RISC-mediated cleavage of
the target RNA, so a U is preferred at position 10. In practice,
these guidelines are a useful starting place, but an
empirical approach is required. Several companies (e.g.,
Imgenex, Ambion, Invitrogen) have developed plasmid vectors
that permit the cost-efficient generation of siRNA by
in vitro transcription. This allows the investigator to use
standard transfection methods to walk down the mRNA and
identify which potential siRNA work best. At that point,
chemically synthesized duplex RNA can be bought or an
RNA hairpin cloned in a viral vector. The substantial commercial
support and user support for this technology makes
RNAi accessible to any investigator with tissue culture
skills.
Pitfalls of RNAi
The problems with RNAi have been widely discussed by its
practitioners. These generally fall under the heading ‘‘off
target’’ effects. Such effects have several possible sources,
including induction of the interferon response, incorporation
of the sense strand into RISC, and the cleavage of an RNA
with partial sequence identity to the intended target. As few
as eight or nine consecutive base pairs at the 50 terminus of
RISC-associated RNA is sufficient to mediate cleavage.
Consequently, some have proposed using microarray analysis
in association with each siRNA experiment to monitor
potential off-target effects. A more practical approach, and
one that applies to the other antisense technologies, is to
employ two or more siRNA (ODN, ribozymes) for each target
mRNA and to determine if the same concentration-dependent
physiological consequences are observed. If two inhibitors
of different sequence composition have identical
results, then off-target events are a less likely explanation.
Another way to avoid inducing non-specific effects with si-
RNA is to screen for the most potent inhibitors. Those that
lead to substantial mRNA reduction at low nanomolar concentrations
are less likely to lead to secondary effects by
blocking normal miRNA-mediated processes. Because
Dicer and RISC may be saturable, it is better to use
one highly active inhibitor than to combine a series of
mediocre ones.
56 LEWIN ET AL JID SYMPOSIUM PROCEEDINGS
Delivery of Genes as Nucleotides
The stratum corneum acts as the major barrier to penetration
of molecules across the skin, and large negatively
charged molecules such as antisense ODN, TFO, or siRNA
will not easily penetrate it (Wraight and White, 2001). Topical
application of simple aqueous solutions of oligonucleotides
results in little penetration into the viable epidermis of human
skin. Transfection of the skin using plasmids can be
achieved by compromising the stratum corneum using intense
brushing, adhesive glue stripping, or chemical depiliation.
Specific formulations of nucleic acids including
nanoparticles (Cui and Mumper, 2002) and lipid vesicles
(Baca-Estrada et al, 2000) do increase their transfer to
deeper layers of the skin (Maruyama et al, 2001; Babiuk
et al, 2002; Prokop et al, 2002; Raghavachari and Fahl,
2002), but transfer is most efficient in hair follicles or in sites
of wounding. Ironically, cationic phospholipids used to improve
DNA delivery to cultured cells make delivery less efficient
to skin in vivo.
As a consequence, techniques such as electorporation,
microinjection, and particle bombardment have been tested
in animals and in people (Maruyama et al, 2001; Meuli et al,
2001; Peachman et al, 2003). Oligonucleotide administration
thus remains a critical area for research. Although
antisense ODN, TFO and stabilized siRNA all have tremendous
potential as topical agents for blocking the expression
of dominant-negative keratin proteins, their exploitation demands
a safe and tolerable delivery system. As it can be
discontinued, topical delivery of therapeutic nucleotides is
inherently safer than viral-mediated gene therapy. For
this reason, and because it will require life-long administration,
this approach will be attractive to the pharmaceutical
industry.
Viral Gene Delivery
Viruses have been selected by nature for gene delivery to
cells. For this reason viral vectors provide the most effective
delivery vehicles for transmitting therapeutic genes into the
body. Vectors based on adenovirus type 5 (Ad5) are efficient
at gene delivery to the skin, and despite their inflammatory
and immunogenic properties, can lead to expression of
therapeutic genes over a period of weeks in wounded epidermis
(Doukas et al, 2001; Gu et al, 2004). AAV, which like
adenovirus can infect both dividing and quiescent cells, will
also infect keratinocytes, and wild-type AAV will replicate in
a helper independent fashion in differentiating cells (Meyers
et al, 2000; Galeano et al, 2003). But in the absence of the
viral rep protein, AAV vectors do not usually integrate into
chromosomal DNA and are diluted out of replicating cells
in vivo. They may therefore be unsuitable for long-term
transduction of the epidermis (but see Hengge and Mirmohammadsadegh,
2000). Repetitive administration of
Ad5 or AAV is precluded by the immune response to the
vector.
For these reasons, retroviral vectors are the best current
delivery system for long-term expression of therapeutic
genes in the epidermis. Retroviral vectors integrate, express
exogenous genes and provide an efficient transfer tool for
human gene therapy applications (Mulligan, 1993). Vectors
based on murine leukemia virus (MLV) have been used for
transduction of epidermal skin cells in vivo (Ghazizadeh
et al, 1999). Transcription in these vectors is dependent on
the viral promoter/enhancer in the 50 viral long terminal repeat
(LTR), permitting constitutive expression of the therapeutic
gene in keratinocytes. But as mentioned above, for
diseases like PC, which affect the upper layers of the skin, if
genes are transferred to stem cells, it might be important to
keep therapeutic genes silent until these cells differentiate.
Otherwise, keratin isoforms may be inappropriately expressed
in the basal layers of the epidermis and stem cells
may have reduced capacity for self-renewal. Although attempts
have been made to obtain tissue specificity by inserting
a tissue-specific promoter internally within retroviral
vectors or by deleting viral enhancer elements, these approaches
frequently lower viral titers and fail to achieve cell
type specificity. Ghazizadeh et al (2002) achieved targeted
gene expression in a stratum-specific manner in the epidermis
using the upstream regulatory elements of the human
involucrin gene to replace the viral enhancer element in
the U3 region of the LTR. Involucrin is a precursor protein
used to assemble the cornified cell envelope and is expressed
in the suprabasal layers of the stratifying epithelia.
Ghazizadeh et al achieved good viral titers (2 106 per mL)
Figure 7
Guidelines for design of siRNA. See
Mittal (2004).
10 : 1 OCTOBER 2005 GENE THERAPY OF KERATIN DISORDERS 57
and demonstrated expression of a marker gene (GFP) in
epidermal cell lines and in the suprabasal layers of raft cultures
of human keratinocytes. Infection of mouse skin with
this vector led to long-term (20 wk) stratum-specific expression
of GFP in the mouse epidermis.
HIV-1-derived lentiviral vectors can efficiently infect
slowly dividing and non-dividing cells, including stem cells
(Rubinson et al, 2003), and demonstrate more stable expression
than MLV-derived vectors (Zaiss et al, 2002). In
order to reduce the risk of contaminating preparations of
recombinant virus with infectious HIV-1, helper plasmids
contain deletions for many genes required for productive
infection. In addition, current lentiviral vectors are ‘‘self-inactivating’’;
they contain deletions of promoter elements in
the 30 LTR that become incorporated into the 50 LTR upon
replication. At least one lentivirus vector has been approved
for use in human therapy (VRX496 from VIRxSYS of Gaithersburg,
Maryland) Lentivirus vectors have been used
successfully for long-term gene delivery to the skin (Baek
et al, 2001). Although gene therapy vectors based on feline
immunodeficiency virus and other non-primate lentiviruses
are being developed (Poeschla, 2003), these are currently of
lower infectious titer than HIV-1-based viruses and they
have not been demonstrated to be safer for use in humans.
In a recent and exciting application of lentiviral gene delivery
to the skin, Siprashvili and Khavari (2004) generated a
series of vectors in which gene expression and persistence
in the skin are regulated by the administration of steroid
hormones. To control the expression of the transferred gene
they used promoters containing a series of glucocorticoid
response elements. To control the persistence of the provirus
elements in the skin, they included loxP sites in the
LTR and genes for a Cre-estrogen receptor fusion protein in
some of the vectors. Upon activation of the fusion protein
with a 4-hydroxytamoxifen cream, most of the provirus was
excised from host genomic DNA, leaving an inactive LTR
behind. These investigators used a single intradermal injection
to deliver a recombinant virus encoding the Epo
gene with a view to using the skin as a depot for the systemic
delivery of proteins. Upon topical glucocorticoid induction,
a rapid increase in hematocrit was observed,
indicating a therapeutic effect. More importantly, when the
animals were allowed to recover for 30 d and the hematocrit
returned to basal levels, a second round of stimulation led
to a similar increase in plasma Epo and hematocrit was
obtained, indicating stable introduction of a regulated genetic
element.
Non-Viral Delivery of Genes
The major compartments comprising the skin are easily
isolated, expanded in vitro and grafted back to the donor
following therapeutic manipulation. This is the basis for the
epithelial transplants currently used in the treatment of
burns. Such modified epithelia can be monitored and removed
if cessation of treatment is indicated. Therefore, direct
delivery of plasmid DNA encoding therapeutic genes
may be possible ex vivo followed by autologous transplant
to the patient. To achieve stability, such genes must be delivered
to stem cells and stably expressed, implying integration
into active chromatin of the patient’s DNA. Several
of these non-viral technologies, including retrotransposition
(e.g., the Sleeping Beauty transposon) and rare cutting
bacterial integrases (e.g., fC31) have been piloted for use
in inherited skin disease (Ortiz-Urda et al, 2002, 2003). This
approach would be useful for the long-term delivery of
several of the therapeutic genes described above, including
zinc-finger repressors, and genes encoding ribozyme
or shRNA. In addition, cultured keratinocytes and their
progenitor cells are ideal for gene correction or gene ablation
strategies using oligonucleotides, zinc-finger recombinases,
or TFO. Cells containing a correction or disruption
of the disease-causing allele could be identified and then
expanded. But ex vivo gene transfer followed by tissue expansion
and grafting will be expensive and time consuming.
In addition, grafting of genetically modified cells is based on
the assumption that the processes of adhesion, growth and
differentiation of keratinocytes will not be modified by the
expression of the transferred gene. As these processes are
strictly regulated by the sequential expression of a series of
genes from the basal to the upper layers of the epidermis,
the therapeutic gene must not interfere with this highly regulated
developmental program of proliferation and differentiation
(Ghazizadeh et al, 2002).
Animal Models of Keratin Diseases
Developing animal models for PC and other keratin disorders
is the subject of another paper in these Symposium
Proceedings. Such models are critical for understanding the
pathology of rare diseases and for testing the efficacy and
safety of therapies (Coulombe and Omary, 2002; Porter and
Lane, 2003). Models comprised of human skin grafted onto
immune deficient mice have been useful in reproducing the
pathological effects of mutation and determining whether
these can be corrected by gene transfer. These models do
not permit study of the immune responses to therapy, which
are critical factors for treatment of homozygous recessive
diseases, and are also important when using viruses or even
naked DNA to restrain expression of a dominant disease
gene.
Mouse models in which disease genes have been disrupted
may be useful for mimicking recessive loss-of-function
mutations (Wojcik et al, 2001; McGowan et al, 2002),
but such models cannot recapitulate disease associated
with dominant-negative missense mutations (Cao et al,
2001). For these, transgenic or knockin models are required.
For testing therapies that depend on nucleotide sequence
the genotype as well as phenotype must be duplicated.
From the genetic standpoint, it might be more
interesting to produce the human mutation within the context
of the homologous mouse keratin gene (if that can be
identified). Constructing such a knockin model permits detailed
investigation of the pathophysiology of the disease,
using a mutated gene that is appropriately regulated at its
normal chromosomal locus. For the gene therapist trying to
develop an antisense therapy or to target gene correction,
such a mouse model provides a useful proof-of-concept
test. Nevertheless, to test the efficacy of a therapy to
be used in humans, the human disease gene must be
58 LEWIN ET AL JID SYMPOSIUM PROCEEDINGS
corrected or suppressed. For a valid test of these nucleotide
sequence-specific therapies, a human disease gene (as
a knockin or a transgene) must be the cause of pathology.
Only then can the tool to be used in people be tested.
Conclusions
There are several clear and plausible pathways to successful
gene therapy for diseases caused by dominant keratin
mutations. The obstacles along these pathways may be
economic as well as scientific: given the rarity of these diseases,
they will not become targets for pharmaceutical
companies or for venture capitalists who fund much of the
translational research in biotechnology. Therefore, the best
line of attack, targeted gene correction, may not be the
most practical. Given the accessibility of the skin, antisense
oligonucleotides, triplex-forming molecules, or RNA interference
using synthetic and stabilized nucleic acids is the
most attractive approach. Success of these methods depends
on the ability of pharmacologists and dermatologists
to devise painless and consistent delivery methods for
oligonucleotide medicines. This problem has been recognized
for some time.
DOI: 10.1111/j.1087-0024.2005.10207.x
Manuscript received June 9, 2005; revised June 27, 2005; accepted for
publication June 28, 2005
Address correspondence to: Alfred S. Lewin, University of Florida, Box
100266, Gainesville, Florida 32610-0266, USA. Email: lewin@ufl.edu
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