CHAPTER 1
Introduction

2In 1990, the first genetically modified cells administered to humans occurred
when retroviral vectors were used to mark tumor-infiltrating lymphocytes (1). This was
shortly followed by the first infusion of modified cells for the correction of a genetic
disease in a four-year old girl with adenosine deaminase deficiency (ADA-SCID) (2).
Since then, despite major advances in gene delivery to human cells, even the most
successful clinical trials have used methods that rely upon quasi-random integration of
therapeutic DNA (3). With the publication of the human genome draft in 2000, its
continued refinement and the annotation of genomes from a vast number of other
species, we now have a concept of where we should be targeting genetic insertions.
Using this knowledge, we can now engineer either viral or non-viral gene delivery
vehicles to predetermined sites that are deemed safe and capable of continued long-term
transgene expression. By engineering site-specific gene targeting vectors, numerous
fields in both basic and applied research would be greatly benefited.
This thesis documents research performed to engineer site-specific gene
targeting in the human genome and its application in hematopoietic stem cells. This
chapter outlines the current methods available for integrating transgenic material and
progress in site-specific gene targeting.
Integrating viruses
To date, the most successful and widely used gene therapy applications have
utilized retroviruses. The family of retroviruses, including the lentivirus, has thus far
been the sole proven means to cure human immunogenic diseases. Two ongoing trials
in France and the U.K. have successfully cured X-linked SCID in 17 out of 20 enrolled
children. Equally as successful has been gene therapy for ADA-SCID, which has seen
more than 10 children cured who were enrolled in separate British (4) and Italian (5)
trials. Despite this success, it has come at a cost, as four out of the 10 boys in the X-
linked SCID trial went on to develop T-cell leukemia, resulting in one death. The three

3remaining boys are currently undergoing leukemia treatment that appears to be working.
Once thought to be completely random in its integration profile, lentiviruses have now
been shown to preferentially insert transgenes into actively transcribed regions (3).
More damaging to the case for retroviruses is that these integration events have a
propensity for 5’-end insertions (6), which amplifies the risk for proto-oncogene
stimulation. Such was the result in the X-SCID trials, where at least two of the affected
boys had insertional mutagenesis at the 5’-end of the LMO2 gene (7).
Another family of viruses, encompassing the adeno-associated viruses (AAV), is
capable of modest site-specific integration into chromosome 19 when either Rep68 or
Rep78 encoding genes remain in the encapsulated viral genome (reviewed in 8). There
are two major drawbacks to using recombinant AAV for the purpose of gene therapy.
The first is that the viral payload is limited to 5kb. This presents a serious problem
when Rep78 or Rep68 must be present, in addition to a donor gene, for site-specific
integration. To alleviate this size restriction, recombinant AAV using two different
AAV vectors has been employed (9). Unfortunately, these also demonstrated a
preference for integration into active genes. The second drawback is that most people
are seropositive for a number of the currently available AAV strains for gene therapy,
which poses an immunogenicity risk (8, 10).
There has been an ongoing attempt to alter retrovirus targeting to predetermined
sites. By fusing Zif268 (11), LexA (12) or designed polydactyl zinc finger E2C (13)
DNA binding domains to either the C-terminal or N-terminal domain of HIV-1-IN,
several labs have reported site-specific integration, although at extremely low
efficiencies. And in perhaps what may become the future of site-specific targeting,
Naldini and colleagues designed a zinc finger nuclease (ZFNs) in tandem with an
integrase-defective lentivirus to stimulate homologous recombination at the IL-2
receptor common gamma-chain gene. They were able to demonstrate gene addition in
human cell lines at high levels (~50%) and in human ESCs at ~5% (14). A similar

4system with zinc finger binding domains is demonstrated with phiC31 integrase in
chapter 4 of this thesis.
While ZFNs appear to be a promising recombinant strategy for engineering viral
site-specificity, the additional disadvantages of large-scale production costs for any of
the above viral systems may be too burdensome in the long run. This would limit their
utility to lab research and gene targeting in non-clinical industries that do not require the
strict production standards of human gene therapy. Aside from ex vivo strategies, many
viruses pose an immunological risk as briefly mentioned above in regards to AAV. Two
dramatic cases, one involving adenovirus (15) and another using AAV (16) resulted in
immune complications leading to death, although, the exact cause of the AAV gene
therapy death is still being debated.
While virus-based gene therapy has shown some success, researchers are
starting to pay closer attention to non-viral alternatives that avoid many of the problems
cited above.
Homologous recombination and zinc finger nucleases
For years, the use of homologous recombination (HR) has been limited to events
for which, selection could be employed or when screening for thousands of potential
recombinants was feasible. This is due to the rare event of HR that can recombine
exogenous DNA with homology to genomic DNA at frequencies of only 10
-6
(17). This
means that when introducing a transgene, HR will be a rare event in comparison to non-
homologous end joining, which can randomly integrate the same transgene at nearly
1000-fold higher frequencies (17). By introducing double stranded breaks (DSBs) with
endonucleases, HR can be increased more than 10-fold (18).
A more powerful solution has been demonstrated by using zinc finger nucleases
that can create site-specific DSBs. These nucleases are synthesized to include an
endonuclease, most commonly FokI, fused to the N-terminus of a DNA binding zinc

5finger. These zinc finger domains can be designed to target, in theory, any location in a
genome. The use of zinc finger nucleases has dramatically increased the rate of DSBs,
and therefore HR, by several thousand-fold (19). Zinc finger motifs are modular in
structure, and therefore can be swapped with other fingers without destroying the
overall backbone. Each zinc finger binds to three nucleotides and multiple fingers can
co-exist on the same fusion product. By adding just three or four fingers then, ZFNs can
be designed to target highly specific sequences of 9 or 12 bases. By introducing a
heterologous mix of two such ZFN constructs, a precisely targeted DSB and HR can be
stimulated. This is a fairly nascent technology, which currently suffers from the
inducement of cytotoxicity primarily due to off-target DSBs. In chapter 4 of this thesis I
demonstrate the use of a zinc finger strategy coupled with the phiC31 integrase, which
may reduce off-target events. Others have utilized lentiviral vectors to shuttle ZFNs into
cells as mentioned previously. And lastly, resolvases and invertases have each been
fused to ZFNs in lieu of the FokI endonuclease (20, 21).
Transposons
Several transposon systems have demonstrated some utility in site-specific
integration. Transposons are able to mobilize themselves around genomes through a
“cut and paste” mechanism. These examples include Sleeping Beauty (22), Tol2 (23),
Frog Prince (24) and piggyBac (25). Each of these transposon systems integrates
transgenes through the use of a transposase that binds to short inverted repeats at the
ends of the transposon to be integrated. Sleeping Beauty (SB) exclusively targets TA
dinucleotides. Due to the high number of such TA dinucleotides throughout a genome,
SB targets thousands of potential integration sites. Because of this, SB has been widely
adopted for research involving the identification of novel oncogenic elements in various
cells including mouse ESCs (26). Despite SB’s insertional mutagenesis profile, it has

6been used for therapeutic gene targeting in mouse liver (27), mouse lung, human
primary T-cells (28), and even in human embryonic stem cells (29).
The utility to each of the above organism and tissue targets is limited due to the
nearly ubiquitous distribution of the SB preferred TA dinucleotides. To improve the
site-specificity and efficiency of SB, DNA binding domains fused to the transposase
have been used, including a zinc finger DNA binding domain. While these chimeras
were able to decrease random integration events to an extent, the efficiency of
transposon-mediated integration was dramatically reduced (30). And while the
piggyBac transposon has been shown to retain efficiency when fused to a GAL4 DNA-
binding domain (25), there are reports that undesired transpositional hopping occurs
(31). Finally, these transposon systems are limited in their capacity to integrate sizeable
transgenes, as efficiency is inversely related to the size of the exogenous DNA (32).
Site-specific recombinases
The serine recombinase family and the related tyrosine recombinases are site-
specific enzymes with diverse capabilities in terms of recombination mechanisms and
requirements. The tyrosine family, so named because of their catalytic tyrosine,
contains the well-known Cre and Flp recombinases. Cre protein is capable of
recombining two identical loxP sites. LoxP sites are 34 bases in length and consist of an
asymmetric 8 base-pair core flanked by two 13 base pair palindromic stretches. Flp
protein is analogous to Cre. It recombines two identical FRT sites, which are 34 bases in
length, including an 8 base core. While both Cre and Flp are efficient at catalyzing the
recombination between their respective DNA recognition sites, LoxP and FRT, those
sites do not natively exist in the human genome. For practical gene therapy, an
alternative is to evolve Cre or Flp towards a predetermined site (33, 34).
A major limitation of Cre and Flp is that they are bidirectional, meaning
excision is at least as likely to occur as integration because of the identical nature of the

7recombination sites (35). Distantly related to the tyrosine recombinase family are the
serine recombinases, characterized by their catalytic serine. The serine recombinases are
highly efficient at catalyzing recombination, in part because of the four-strand break
mechanism, which they employ. This catalysis is in concert with covalent binding of the
DNA to the recombinase, which is mediated by the active serine. Two classes within the
serine recombinase family, the invertases and resolvases, are bidirectional. These
include the resolvase, Tn3 resolvase, and the Hin and Gin invertases. Like Cre and
Flp, these smaller recombinases recombine two identical sites. So, while they are site-
specific, the invertases and resolvases from both the tyrosine and serine recombinase
families would require modification to prevent excision in order to be useful gene
therapy tools. In contrast, a third class within the serine recombinase family, known as
the integrases, is capable of unidirectional site-specific recombination between two non-
identical att sites, attB and attP. The recombination between attB and attP sites carried
out by integrases results in hybrid sites termed attL and attR (36). These new att sites
are not recognized appreciably by the serine integrases, thus making them refractory to
the back reaction.
Amongst the unidirectional site-specific serine recombinases is an integrase
from the bacteriophage phiC31. The integrase was originally found to mediate site-
specific recombination of its phage genome into that of its Streptomyces host genome
(37). In its native bacterial context, phiC31 will recombine its attP site with the
bacterial attB site as illustrated in figure 1-1. Both attB and attP share an identical two
base core of TT at which the recombination crossover occurs. Flanking the TT core is a
set of non-identical inverted repeats. The minimal attB and attP sizes that can be
effectively recombined have been found to be 34 and 39 base pairs, respectively (38).
My lab was the first to show that the phiC31 integrase is capable of site-specific
recombination in mammalian cells. Various mammalian genomes have been shown to
possess sites similar to the phage attP site, termed “pseudo attP.” Placing the reciprocal

8attB site onto a donor plasmid and co-delivering a plasmid encoding the phiC31
integrase can recombine the attB bearing plasmid with pseudo sites (38). This reaction
occurs at an efficiency of 5-10%, requires no host co-factors, has demonstrated
recombination in a range of organisms, and its site-specific integration stands in
contrast to viral vectors in current use. Finally, a large phiC31 integration profile study
has shown that ~56% of all integration events in human cells occur in just 19 “hotspots”
(39).
In the work here, I demonstrated that while many large serine integrase family
members have been discovered recently, phiC31 still out performs them in terms of
recombination efficiency (Chapter 2). I have also shown that phiC31 is capable of
mediating stable transgene expression in lineage depleted mouse hematopoietic stem
cells (Chapter 3). We estimate that there are approximately 370 accessible phiC31
pseudo sites in the human genome (39). Compared to viral integration profiles, this
number is several orders of magnitude lower. Still, many of the current phiC31
integration sites are undesired because of either safety concerns or suppressed
transcriptional activity. To address these issues, I have demonstrated the ability to fuse a
zinc finger DNA binding domain to the catalytic domain of phiC31 to alter its
specificity (Chapter 4). I have also begun using directed evolution strategies to engineer
a phiC31 derivative with increased recombination frequencies toward one of the
hotspots, on human chromosome Xq22.1 (Chapter 5). The work shown in this thesis has
provided new insights into phiC31 enzyme function and augmented the options
available for site-specific gene targeting.

9Figure 1-1. Schematic diagram of phiC31 recombination. A. In nature, the phiC31
bacteriophage attP site has partial similarity to a DNA sequence, termed attB, in the
Streptomyces genome. phiC31 integrase catalyzes the integration of attP into attB,
thereby inserting the phage genome into the Streptomyces genome. B. Mammalian
genomes have partially matching sequences to attP, termed “pseudo attP sites.” A
donor plasmid bearing the attB site co-delivered with phiC31 integraese will integrate
into the chromosome. This reaction is unidirectional, as the original att site recognition
sequence is destroyed with the creation of hybrid attL and attR sites.

10
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