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Improving adeno-associated viral vector for hematopoietic stem cells gene therapy

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Improving adeno-associated viral vector for hematopoietic stem cells gene therapy

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Content IMPROVING ADENO-ASSOCIATED VIRAL VECTOR FOR
HEMATOPOIETIC STEM CELLS GENE THERAPY

By

Hsu-Yu Chen

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)

May 2016

Copyright 2016 Hsu-Yu Chen

Table of Contents
Acknowledgements!............................................................................................................................!i!
List of Figures!...................................................................................................................................!ii!
Abstract!.............................................................................................................................................!1!
Chapter 1.! Introduction!..............................................................................................................!3!
1.1! Gene therapy!..................................................................................................................................!3!
1.2! Hematopoietic stem cells!...............................................................................................................!4!
1.3! Adeno-associated viral vector!.......................................................................................................!7!
1.4! Heparan sulfate binding mutation!...............................................................................................!9!
1.5! Proteasome activity!.....................................................................................................................!10!
Chapter 2.! Materials and Method!...........................................................................................!14!
2.1! Cell culture!...................................................................................................................................!14!
2.2! Recombinant AAV Vectors production!.....................................................................................!14!
2.3! Mutant cloning!............................................................................................................................!15!
2.4! Heparin binding affinity of mutant virus!..................................................................................!15!
2.5! AAV Transduction and proteasome inhibitor administration!................................................!16!
2.6! FACS!............................................................................................................................................!16!
2.7! Genomic DNA extraction!............................................................................................................!17!
2.8! Colony forming unit (CFU) assay!..............................................................................................!17!
2.9! Statistics!.......................................................................................................................................!17!
Chapter 3.! Results!.....................................................................................................................!18!
3.1! Examine the potential of heparin binding-deficient AAV6 as liver de-targeting vector and
enhance transduction in HSC!..................................................................................................................!18!
3.1.1! K531E mutant showed reduced heparin binding affinity!.......................................................!18!
3.1.2! K531E mutation reduce transduction in liver cells and HSC!.................................................!19!
3.2! Enhance transduction by preventing proteasome degradation during intracellular
trafficking!.................................................................................................................................................!20!
3.2.1! Double and triple tyrosine mutation has no positive effect on HeLa or HSC transduction!..!20!
3.2.2! Proteasome inhibitor LLnL enhance gene delivery by AAV6 in HeLa cells!..........................!21!
3.2.3! Proteasome inhibitor LLnL enhance gene disruption by AAV6 delivered in HeLa cells!......!23!
3.2.4! Proteasome inhibitor LLnL toxicity on HSC!..........................................................................!23!
3.2.5! Proteasome inhibitor LLnL increase AAV6 delivered GFP expression in HSC!....................!24!
3.2.6! LLnL administration affect HSC proliferation and differentiation!.......................................!24!
Chapter 4.! Discussion!................................................................................................................!26!
4.1! The potential of heparin binding-deficient AAV6 as liver de-targeting vector and enhance
transduction in HSC.................................................................................................................................!26!
4.2! Tyrosine mutation on AAV6 capsid has no positive effect on HeLa or HSC transduction!...!28!
4.3! Proteasome inhibitor LLnL increase gene transfer and disruption by AAV6 gene delivery in
HeLa and HSC!..........................................................................................................................................!29!
Figures!.............................................................................................................................................!34!
Figure 3. Heparin elution profiles for AAV6 and K531E mutant.!.............................................................!34!
Figure 4. Transduction comparison of AAV6 and K531E mutant in HeLa, HepG2 and HSC.!.................!35!
Figure 5. tyrosine mutant transduction in HeLa and HSC.!.........................................................................!37!
Figure 6. Proteasome inhibitor increased viral DNA accumulation in HeLa cells transduced with AAV6-
GFP.!...........................................................................................................................................................!38!
Figure 7. LLnL increased GFP expression with AAV6 transduction in HeLa cells.!..................................!39!
Figure 8. Proteasome inhibitor increased viral DNA accumulation in HeLa cells transduced with AAV6-
SaCas9.!.......................................................................................................................................................!40!
Figure 9. LLnL increased gene disruption by AAV6 delivered CCR5 targeted SaCas9 in HeLa cells.!.....!41!
Figure 10. Cell toxicity analysis of LLnL treatment on HSC.!....................................................................!42!
Figure 11. LLnL increased AAV6 delivered GFP expression in HSC.!......................................................!43!
Figure 12. LLnL administration affect HSC proliferation and differentiation.!..........................................!44!
Appendix!.........................................................................................................................................!45!
Modified AAV production protocol for Cannon Lab!...........................................................................!45!
References!.......................................................................................................................................!54!
!

i! !
Acknowledgements
I would like to thank Dr. Paula Cannon for giving me the opportunity to join this wonderful lab and
learn all these important research topic and skills. Dr. Cannon has been a great advisor. Her
guidance and support along the way of my learning process have been invaluable.
I would also like to thank all lab members for ideas discussion and suggestions that help me to
improve my presentation. I would like to thank especially Cathy Wang for helping me edited my
thesis, Morgan Chateau, Jessica Rathbun and Cathy Wang again for all the advises that help me
establishing AAV production in our lab, Eduardo Seclén for saCas9 mediated gene disruption
analysis and the all the guidance.
Finally, I would like to thank Dr. Shou-Jiang Gao and Dr. I-Chueh Huang for dedicate their time
being my thesis committee members and for all the critics and suggestion they provide to help me
improve my thesis.

ii! !
List of Figures
!

Figure 1. cell receptors for AAV attachment expressed on different cell surface
and the transduction efficiency of K531E mutant in different cell line. 10

Figure 2. ssAAV and scAAV genome replication. 12

Figure 3. Heparin elution profiles for AAV6 and K531E mutant. 34

Figure 4. Transduction comparison of AAV6 and K531E mutant in HeLa, HepG2
and HSC. 35

Figure 5. tyrosine mutant transduction in HeLa and HSC. 36

Figure 6. Proteasome inhibitor increased viral DNA accumulation in HeLa cells
transduced with AAV6-GFP. 38
!
Figure 7. LLnL increased GFP expression with AAV6 transduction in HeLa cells. 39
!
Figure 8. Proteasome inhibitor increased viral DNA accumulation in HeLa cells
transduced with AAV6-SaCas9. 40
!
Figure 9. LLnL increased gene disruption by AAV6 delivered CCR5 targeted SaCas9
in HeLa cells. 41

Figure 10. Cell toxicity analysis of LLnL treatment on HSC. 42

Figure 11. LLnL increased AAV6 delivered GFP expression in HSC. 43

Figure 12. LLnL administration affect HSC proliferation and differentiation. 44!
1! !
Abstract
Genome editing in hematopoietic stem cells (HSC) through ex vivo or in vivo approaches are
promising strategies for curing several blood cell related diseases such as HIV infection. However,
delivery of genome editing reagents has proven to be challenging in HSC in both approaches.
Ex vivo genome editing by Electroporation of ZFN mRNA, which is the current method of choice
for ex vivo HSC genome editing, results in ~5% transgene insertion, but the engraftment data
reveal that it is still challenging to modify the most primitive HSC. Our previous study using
AAV6 as a donor delivery vector successfully achieve high genome editing results (23%) in the
most primitive HSC population indicating a promising editing strategy using AAV6.
However, there are two potential disadvantages to the ex vivo approach, First, the isolation and
culture of HSC carry some risk of contamination. Second, the ex vivo culture of HSC can reduce
their stem cell function. For these reason, an in vivo approach is more desirable. The challenges
of using viral vector for in vivo genome editing to date are the low transduction efficiency in vivo,
the genome integrating issue and the immunogenicity. Adeno-associated viral (AAV) vector is
also an ideal system for in vivo editing purpose, due to their ability to transduce quiescent cells,
their transient expression profile upon cell proliferation, and their stability and low
immunogenicity in vivo.
Despite all the advantages, there are still difficulties for AAV-mediated delivery of genome editing
reagents to HSC. AAV transduction efficiency in HSC is relative low compared to that in cell
lines. Furthermore, in vivo HSC transduction efficiency is further lowered due to broad tissue
2! !
tropism of AAV and the preferential transduction in the liver, causing loss of vectors in targeted
tissues.
In this study, we explore potential strategies to enhance in vivo transduction of HSC by AAV
vectors by 1) de-targeting AAV vectors from liver cells to increase their availability for HSC
transduction in vivo, and 2) improving intracellular trafficking of AAV vectors to increase
transgene expression. To promote liver de-targeting, we use AAV vectors carrying capsid proteins
with one mutation, K531E, which has been shown to function in AAV binding to liver cells but
has an unknown role in binding to HSC. To improve intracellular trafficking and transgene
expression, we use capsid tyrosine mutations and proteasome inhibitor treatment to allow AAV
vectors to overcome proteasome-mediated degradation.
In the results, we found that the K531E mutant AAV demonstrated a 10-fold decrease in
transduction of a liver cell line, with a concomitant 3-fold decrease in transduction of HSC. While
these findings support the liver de-targeting activity of the K531E mutant, the decreased HSC
transduction limits the usefulness of this AAV for delivery of genome editing reagents to HSC in
vivo. In addition, we found that proteasome inhibitor significantly increased AAV transduction in
HSC. Whether the functionality of primitive HSC is preserved after proteasome inhibitor treatment
remains to be determined.
3! !
Chapter 1.!Introduction
1.1!Gene therapy
Gene therapy is a therapeutic method which introduces a foreigner gene into the target
cells. The target cells could be infected cells or has gene deficiency such as ADA-SCID
(Aiuti et al., 2002), X-linked adrenoleukodystrophy (Cartier et al., 2009) or HIV infection
(Hoxie & June, 2012). The transgene can play a role in functional protein expression or
disrupt/correct the genomic DNA (genome editing) in targeted cell.
To do the gene editing, targeted nucleases such as zinc-finger nucleases (ZFNs) and RNA-
guided clustered regulatory interspace short palindromic repeat (CRISPR)/Cas
endonucleases are powerful tools. The nucleases function by creating site-specific DNA
double-strand breaks (DSBs) (Gaj, Gersbach, & Barbas, 2013), which are repaired by
either non-homologous end joining (NHEJ) or homology-directed repair (HDR) (Wyman
& Kanaar, 2006). The highly error-prone NHEJ DNA repair pathway often results in gene
disruption. HDR utilizes a homologous template for accurate repair of the DNA damage
site. If a modified donor template is introduced, HDR can be used for site-specific insertion
of a desired mutation or gene.
Compared to traditional treatment, gene therapy provides the modification to patients’
genomic DNA and has long-term effect instead of taking traditional drugs repeatedly. For
instance, the active antiretroviral therapy (HAART) for HIV infection required life-long
treatment. The treatment can suppress virus replication, and the viral load can reach
undetectable level (DA, 2000) with a variable increase in CD4 T-cell counts (Valdez et al.,
2002). However, once HAART is discontinued, the replicating HIV reappears in plasma.
4! !
The relapse of the viral load immediately after the treatment stops indicating that the long-
lived viral reservoir persists and patients are not cured (Chun & Fauci, 1999). Moreover,
the life-long drug treatment also brings the problems of cumulative toxicities, incomplete
immune restoration, and the emergence of drug-resistant escape mutants (Hoxie & June,
2012). Given these limitations from traditional drug treatment, gene therapy has been an
encouraging development for the goal of generating lifelong antiretroviral therapy and
eliminate latent viral reservoirs. One of genome editing for human disease that closest to
clinical application thus far is for the treatment of R5-tropic HIV-1 infection. In this
specific study, 12 patients under HAART was enrolled. A pair of highly site specific ZFNs
targeting the HIV co-receptor, CCR5, was introduced to the patients’ CD4 T cells ex vivo,
causing a disruption of the gene. Six of the patient were then undergo 12 weeks of the
HAART interruption 4 weeks after infusion of 10 billion autologous CD4 T cells (11 to 28
% of the cells were genetically modified). Safety, immune reconstitution, and viral load
were then assessed. The viral load increased after 4-6 weeks treatment interruption. For
four of the patient who completes the 12 week HAART interruption, the viral load declined
by an average of 1.2 log10 (range, 0.5 to 2.1) from the peak level during the absence of
HAART. However, only one patient who is heterozygous for CCR5 delta32 did not show
an increase in viral load until 6 weeks treatment interruption, and the viral load decreased
after to the undetectable level before reinstitution of HAART (Tebas et al., 2014).
1.2!Hematopoietic stem cells
Hematopoietic stem cells (HSC) reside in the adult red bone marrow. They are capable of
reconstituting the hematopoietic system and giving rise to both the myeloid and lymphoid
lineages. The unique differentiation and expansion properties mark them as promising
5! !
targets for gene therapy. Even when genome editing is successful in a small subset of HSC,
their self-renewal and differentiation properties will result in the future propagation of the
desired mutation (Bank, 2003). Due to these specified capabilities, HSC gene therapy is
increasingly being applied towards the treatment of a range of blood cell-related genetic
diseases (Aiuti et al., 2002)(Cartier et al., 2009).
However, despite all the advantages of using HSC as gene therapy target, there are several
challenges in gene editing of HSC. There are several ways to deliver gene editing reagent
to the cells including nucleic acids (plasmid DNA, mRNA, and oligonucleotides) and
certain viral vectors integrase-defective lentivirus (IDLV), adenovirus, and AAV).
Application for this method is well-established cell lines and some primary cells (Coluccio
et al., 2013; Lombardo et al., 2011). However, gene delivery and editing are especially
challenging in HSC. (Cox, Platt, & Zhang, 2015). Lombardo and the group utilized IDLV
as vector only achieved 0.1% editing (Lombardo et al., 2007). A further strategy combining
ZFN mRNA with IDLV delivered donor templates increase the site-specific insertion of
GFP to ~5% (Genovese et al., 2014a). Moreover, studies for these edited cells engraft into
immune-deficient mice showed the difficulty of editing the most primitive HSC population
(Genovese et al., 2014b; Megan D Hoban et al., 2015).
There are two ways to deliver transgenes to HSC: ex vivo and in vivo. The ex vivo delivery
involves isolating HSC from the patient, modifying them ex vivo, and transplanting back
into the patient. There are two major drawbacks to this process. First, the target cells have
to be capable of maintaining stem cell properties and survive through the ex-vivo
manipulation. Several approaches were applied to expand HSC ex vivo to reach sufficient
amount for adult transplantation. These include optimizing culture conditions by
6! !
combinations of growth factors (C. C. Zhang, Kaba, Lizuka, Huynh, & Lodish, 2008) small
synthetic chemicals (Boitano et al., 2010) or specific proteins including Notch-ligands
(Delaney et al., 2010). However, all of the modifications showed that most of the
proliferating progeny loss regeneratibe potential within few days (Weidner et al., 2013).
Second, the re-engraftment often has a poor efficiency which limited the editing effect.
Although the engraftment can be increased by depleting unedited cells before
transplantation, the process introduces risks to patients (Cox, Platt, & Zhang, 2015). Other
risks like the needs for the conditioning treatment, as well as graft-vs-host-disease
following allogeneic transplant, are also the concern for the ex vivo gene therapy strategy.
On the other hand, scientists have been studying in vivo delivery. Three different system
have been studied for in vivo delivery: modified naked siRNA, viral vectors, and nonviral
vectors (Y. Zhang, Satterlee, & Huang, 2012). Retroviral vector (RV)-mediated gene
delivery previously proved that resulted in modification of HSC through direct intrafemoral
injection into adult mice (McCauslin et al., 2003). Lentiviral vector (LV) was also
developed for HSC transduction in vivo in unconditioned mice (Worsham, Schuesler, von
Kalle, & Pan, 2006). The advantage of using LV over RV is that LV transduces quiescent
stem cells while RV only transduces cells under division. However, the challenge of using
these viral vectors are the low transduction efficiency in vivo and the immunogenicity of
these vectors. Adeno-associated viral (AAV) vector is one of the most promising systems
for delivery due to its low immunogenicity, high safety, non-integrating genome, and
capabilities for long-term expression in non-dividing cells (Kotterman & Schaffer, 2014).
To reach the goal of developing safe, specific targeting and high efficient viral vector
system, AAV delivery system studies is strongly needed.
7! !
1.3!Adeno-associated viral vector
Adeno-associated virus (AAV) belongs to the family Parvoviridae and genus
Dependovirus. It is a small (25 nm) unenveloped virus. The genome of AAV is a single
positive or negative stranded DNA with the size 4.7 k base pair. The wild-type AAV
genome contains rep and cap genes flanked by two inverted terminal hairpin repeats
(ITRs). ITRs are required elements for successful packaging. There are 12 common
serotypes of AAV (AAV1-12). Each of the serotypes has different tissue tropism based on
their capsid sequence and structure affecting cell receptor binding (Balakrishnan &
Jayandharan, 2014; Z. Wu, Asokan, & Samulski, 2006). Previous studies show that among
all the serotypes, AAV6 has the highest transduction in HSC and as such AAV6 has been
used as a viral vector for gene editing in HSC (Song, Kauss, et al., 2013; Wang et al., 2015).
Using AAV6 as the donor deliver vector, Our lab has successfully performed homology-
driven genome editing in HSC by combining ZFNs targeting CCR5 with a homologous
donor to the region, resulting in the insertion of our desired sequences. The results showed
up to 23% gene modification at the CCR5 locus (Wang et al., 2015). This is a major
improvement on previously published results showing a ~5% modification delivering
ZFNs as mRNA and the donor on an IDLV platform (Genovese et al., 2014a; M. D. Hoban
et al., 2015).
However, there are still some challenges using AAV6 as the vector both ex vivo and in
vivo delivery. Ex vivo studies showed that transduction efficiency of AAV6 in mouse
hematopoietic progenitor cells are relatively low when to compare to the effectiveness of
other cell lines like HeLa cells (Ellis et al., 2013). In human HSC, it also required relatively
high multiplicities of infection (MOI) or cevtor genomes (vgs)/cell to transduce
8! !
successfully (Song, Kauss, et al., 2013; Song, Li, et al., 2013; Wang et al., 2015).
Furthermore, AAV has a small and strict genome package capacity. The packaging of
genomes with a larger size (close to or over 4.7 kb) will decrease the transduction efficiency
by ~70% when comparing between 4.5kb and 6.0 kb of the genome size. (Grieger &
Samulski, 2005; Z. Wu, Yang, & Colosi, 2010). In vivo study in mice showed that AAV
has broad tissue tropism. AAV6 can strongly transduce liver, heart, lung, and muscle
(Zincarelli, Soltys, Rengo, & Rabinowitz, 2008). The off target tissue transduction might
decrease the vector titer that correctly target to the HSC and compromise the safety of the
gene therapy. So far, no successful transduction of HSC through in vivo delivery has been
reported. Due to all the challenges mentioned, a way to improve AAV6 regarding targeting
and transduction ability for HSC gene editing is urgently required.
To increase the transduction efficiency of AAV in HSC, the life cycle of the AAV vector
upon entering a host cell must be understood and modified. The life cycle of an AAV vector
includes (1) cell receptor binding, (2) endocytosis mediated cell entry, (3) intracellular
trafficking and translocation to the nucleus, and (4) transgene expression in the nucleus
(Balakrishnan & Jayandharan, 2014). Several of these points can be targeted for enhancing
transduction into the cell. These modifications can include transgene modification to
improve the gene expression in nucleus (Han et al., 2008), capsid alteration to enhance
receptor binding (Song, Kauss, et al., 2013; Z. Wu, Asokan, Grieger, et al., 2006), and drug
treatments tailored to increasing transduction.
In this study, we are going to investigate the potential of (1) using heparin receptor binding
deficient AAV6 as liver de-targeting vector and enhance transduction of HSC in vivo. (2)
9! !
Enhancing transduction in HSC in vitro by capsid modification or drug administration to
manipulate intracellular trafficking.
1.4!Heparan sulfate binding mutation
The tissue tropism of AAV vectors is determined primarily by the interaction of cell surface
receptors and the viral capsid proteins. Several cell surface receptors were reported that
play an important role in AAV6 cell attachment during transduction. These include
Heparan sulfate proteoglycan (HSPG) and 2,6-N/2,3-N sialic acid (SA) (Z. Wu, Asokan,
& Samulski, 2006). The epidermal growth factor receptor (EGFR) was reported function
as a co-receptor and is necessary for AAV6 internalization (Weller et al., 2010). The study
in AAV2 HSPG binding mutation showed that while liver transduction was abolished,
heart transduction was increased ~90%. The different outcome in tissues indicating the
potential of HSPG binding mutant to increase transduction in cells does not require HSPG
for transduction. Mutating the HSPG binding site on the AAV6 capsid was shown to
decrease liver cell transduction both in HepG2 cells and in mice liver with no impact on
HeLa cells transduction (Z. Wu, Asokan, Grieger, et al., 2006). HeLa cells express HSPG
(Mareel, Dragonetti, & Dacremont, 1979), 2,6-N SA, and 2,3-N SA on the cell surface
(Kumari et al., 2007), whereas HepG2 only expresses HSPG (Barth et al., 2003) and 2,6-
N SA (Z. J. Wu, Miller, Agbandje-McKenna, & Samulski, 2006). Like HeLa cells, human
HSC also expresses HSPG (Nasimuzzaman & Persons, 2012), 2,6-N SA, and 2,3-N SA
(Nicholls et al., 2007). However, no transduction profile of HSPG binding mutant AAV6
was reported on HSC (fig. 1).

10! !

Figure 1. cell receptors for AAV attachment expressed on different cell surface and the
transduction efficiency of K531E mutant in different cell line. HSPG: Heparan sulfate
proteoglycan; 2,6/2,3-N SA: 2,6-N/2,3-N sialic acid.

In this study, we are aiming to examine the role of HSPG binding in HSC for AAV6
transduction, and to evaluate the potential of using HSPG binding mutant as liver de-
targeting vector for in vivo HSC transduction.
1.5!Proteasome activity
During the intracellular trafficking process, the viral vector can be phosphorylated upon
escape from the endosome. The phosphorylation leads to ubiquitination and brings the
vector to proteasome machinery (Douar, Poulard, Stockholm, & Danos, 2001). It was
hypothesized that proteasome mediated AAV capsid degradation could be the major reason
for the significant loss of viral particle and genome delivery to the nucleus and cause low
transduction efficiency in some of the cell types (Monahan et al., 2010). Two strategies
have been proposed to enhance transduction by preventing proteasome activity during
intracellular trafficking- tyrosine mutation on the capsid and proteasome inhibitor
administration.
11! !
Phosphorylation of seven tyrosine residues on AAV2 capsid protein was found to play a
role in proteasome degradation. Mutating these tyrosines into phenylalanine individually
showed a decrease of ubiquitination and increase of gene expression in HeLa cells and
mice hepatocyte after transduction (Zhong et al., 2008). Combining two or three mutations
has even bigger impacts on transduction enhancement. Six of the seven tyrosine residues
in the AAV2 capsid are conserved in AAV6. Song and the group examine the effect of
mutating Y445, 705, and 731 on HSC transduction. The results showed that scAAV6
containing Y705F mutation increased transduction in HSC from 11~16% to 35~58%
regarding GFP expression. The improvement of transduction with double mutant
(Y705F+Y731F) was even higher (73%). Mutations on ssAAV6 capsid also increase
transduction. The mCharry expression increase from 1.42% to 35.4% when comparing WT
with Y445F mutation and 17.0% with Y731F mutation. Interestingly, the scAAV6-Y445F
mutant did not show enhancement in transgene expression(Song, Kauss, et al., 2013; Song,
Li, et al., 2013). This conflicting result indicates the tyrosine mutation might have genome
type specific effect.
Self-complementary AAV (scAAV) vectors can be generated by only introduce a genome
that smaller than half size of the wild-type genome (<2.3kb) (D M McCarty, Monahan, &
Samulski, 2001) or generate a mutation on the ITR to prevent nicking at the terminal
resolution site by host DNA polymerase before it is reached by the replication complex
initiated at the other end (fig. 2) (Douglas M McCarty, 2008).
12! !

Figure 2. ssAAV and scAAV genome replication. (Douglas M McCarty, 2008)
It was suggested that although scAAV contain the complementary sequence for the
genome, it is unlikely that the DNA base-paired inside of the capsid due to the interaction
between the viral genome and the inner capsid shell (Chapman & Rossmann, 1995). The
requirement of viral helicase function for packaging also indicates that the DNA is
unwound as it enters the capsid (King, Dubielzig, Grimm, & Kleinschmidt, 2001). It is
likely that the scAAV genome anneals rapidly after uncoating. The annealing step helps
the scAAV circumvented the synthesis of the second strand DNA, which is one of the
limiting factors for AAV transduction. Thus, scAAV was known has higher transduction
ability in certain cell types (Douglas M McCarty, 2008). However, due to the genome
conformation, scAAV has an even more restricted packaging limitation (2.2 kb).
13! !
The alternative strategy to prevent proteasome degradation of incoming AAV vectors is
through proteasome inhibitor (PI) administration. It is believed that PI could directly block
the degradation of the capsids, increase trafficking efficiency, or indirectly improve
genome stability through an alternative mechanism. PI administration has been reported to
increase nuclear accumulation of AAV particles and the vector genome (Hansen, Qing,
Kwon, Mah, & Srivastava, 2000). It has a potential value in the enhancement of AAV
transgene delivery. Furthermore, co-administration of proteasome inhibitor LLnL (N-
acetyl-L-leucyle-L-leucyl-leucinal, also called MG101) with AAV vector containing the
different size of the genome (4675bp-6019bp) showed the larger impact on the larger
genome (Grieger & Samulski, 2005). LLnL works as a peptide aldehyde inhibitor that bind
to the active site of proteolytic enzymes within the proteasome core and reversibly block
its function (Rock et al., 1994). Taken together, PI treatment improves AAV vector
transduction in two major limitations of the system: genome size and intracellular
trafficking. However, the effect of the PI is tissue or cell type specific. For example, in
vivo study showed that PI injection increase transgene expression in liver by 10-fold but
did not affect expression in muscle (Duan, Yue, Yan, Yang, & Engelhardt, 2000). Thus
far, the effect of PI on AAV transduction of HSC is unknown.
The second aim of this study is to enhance the AAV6 transduction on HSC through
preventing proteasome degradation during intracellular trafficking. Two strategies will be
evaluated in this aim. First, generate double (Y705F+Y731F) and triple
(Y445F+Y705F+Y731F) tyrosine mutation AAV6. Second, co-administration LLnL and
AAV6.
14! !
Chapter 2.!Materials and Method
2.1!Cell culture
HeLa, HepG2 cell lines, and HSC were used for in vitro studies. HeLa cells were cultured
in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% fetal bovine
serum and penicillin-streptomycin. HepG2 cells were cultured in Eagle's Minimum
Essential Medium (EMEM), supplemented with 10% fetal bovine serum (FBS) and
penicillin-streptomycin (P/S). HSC were cultured in StemMACS (MiltenyI Biotec)
supplemented with 50 ng/ml each of SCF, Flt3 ligand and TPO (R&D Systems,
Minneapolis, MN), plus 1% PSA). Human HSC were derived from fetal liver. Livers were
obtained from Advanced Bioscience Resources (Alameda, CA) or Novogenix Laboratories
(Los Angeles, CA) as the anonymous waste sample, with the approval of the University of
Southern California’s Institutional Review Board. To isolate single cell suspensions, the
tissues were disrupted physically and incubated in collagenase. The cells were then isolated
by magnetic-activated cell sorting (MACS) (Mitenyi Biotec), as previously described. HSC
were cultured in StemMACS (MiltenyI Biotec) supplemented with 50 ng/ml each of SCF,
Flt3 ligand and TPO (R&D Systems, Minneapolis, MN), plus 1% PSA).
2.2!Recombinant AAV Vectors production
Recombinant AAV were generated in-house; the modified AAV production protocol is
included in the supplementary method. Briefly, AAV6 and mutant were produced by an
adenovirus-free system. 6 x 106 293 AAV cells were seeded in 15 cm dished. 27 ug of
each plasmid for AAV packaging (three plasmids) were transfected into the cells. Cells
were washed with PBS once at 16 hours post transfection and keep culturing in fresh
15! !
DMEM supplied with 10% FBS and P/S until harvest. For vectors carrying GFP expression
transgene, the GFP expression in cells were checked at 24 hours after transfection to verify
the transfection efficiency. Transfected cells were harvest at 72 hours post transfection.
Cell pellet was freeze and thaw at least once at -80. The pellet was then lysed and proceed
to purification by iodixanol gradient method. The purified vectors were then concentrated
and stored at -80 until used. The viral vector genome titer was determined by qPCR and
the infectious titer for GFP-expressing AAVs were examined by HeLa transduction.
2.3!Mutant cloning
Mutants were designed to characterize regions implicated by the structure in heparin-
binding. pRC6 construct from Cell Biolabs was used as the starting point for the K531E
capsid mutation. The triple tyrosine mutant capsid construct (a kindly gift from Cody Lim)
was the starting point for the double tyrosine mutant. Site-Directed Mutagenesis was
performed to generate mutated capsid construct.
2.4!Heparin binding affinity of mutant virus
Heparin-binding affinity was measured using 1 mL HiTrap Heparin HP columns (GE
HealthCare) follow the previous report (Xie, Lerch, Meyer, & Chapman, 2011). Briefly,
the columns were pre-washed with 10 mL 2 M NaCl followed by 10 mL loading buffer
(25 mM Na Hepes, 30 mM NaCl, pH = 7.2–7.4). 5x10
8
vg of the AAV6 and K531E were
diluted in 5 Ml loading buffer. After sample loading, the column was washed with 25 ml
loading buffer. The vectors were then eluted by a NaCl gradient from 100mM to 1M with
a volume of 5mL for each concentration. The wash and elution fractions were collected.
The vector genome in each fraction were extracted and quantified by qPCR.

16! !
2.5!AAV Transduction and proteasome inhibitor administration
HeLa and HepG2 cells were seeded at 5x10
4
cells per well in 48 well plates the day before
transduction. Before transduction, cells were washed with PBS once and replace the
medium with 200 ul DMEM medium without FBS. Cells were then transduced with
indicated MOI. After 24 hours incubation, 200 ul DMEM containing 10 % FBS were added
to the wells. Cells were then harvested at 48 or 72 hours after transduction and subjected
to GFP expression analysis or DNA extraction for qPCR or gene disruption analysis.
HSC were counted after incubating overnight from the isolation. 10
5
cells were seeded into
96 well plates for each condition with and incubated in 100 ul serum free medium for 30
minutes. Different vg/cell AAV were then added to the wells. 24 hours after transduction,
100 ul serum-containing medium were added, and cells were transferred to a 48 well plate.
Cells were harvest at 48 hours after transduction for GFP expression analysis and 72 hours
for genome disruption analysis.
In the proteasome inhibitor experiment, LLnL were co-administrated at the indicated
concentration into the wells together with the AAV at the time of transduction. After 24
hours incubation, cells were washed with PBS once, and the fresh serum-containing
medium was added. Cells were harvest at 48 hours for GFP expression and 72 hours for
genome disruption analysis.
In all HSC transduction, toxicity was examined 24 hours after transduction by 7AAD and
Annexing five staining.
2.6!FACS
Cells were collected at different time points and analyzed for GFP expression by flow
cytometry. A BD FACS Canto II (BD Biosciences, San Jose, CA), or Guava EasyCyte 6-
17! !
2L or EasyCyte 5HT (EMD Millipore, BiLLERICA, MA) were used for GFP analysis.
FlowJo software was used for data analysis. The gating for GFP+ population were set on
the criteria that gives 0.1% or less of the cells from untreated culture being included in the
GFP+ gate.
2.7!Genomic DNA extraction
The genomic DNA from cells were extracted with MasterPure DNA Purification Kit
(Epicentre Biotechnologies) followed manufacturer protocol. Briefly, cells were pelleted
by centrifugation at 1000 rpm for 10 minute. 300 ul of Tissue and Cell Lysis Solution
containing the proteinase K was then added to the cells and mixed thoroughly. After
incubated for 30 minutes, place the samples on ice for 5 minutes. The cell proteins were
then precipitated with MPC Protein Precipitation Reagent, and the DNA was pelleted by
add in isopropanol. 70% Ethanol was used to wash the DNA. Finally, DNA was
resuspended in TE buffer.
2.8!Colony forming unit (CFU) assay
For colony formation assays, the protocol was described previously (Wang et al., 2015).
Cells were plated as a single-cell suspension at a density of 120 cells/ml in semi-solid
methylcellulose-based medium containing 50 ng/ml SCF, 20 ng/ml GM-CSF, 20 ng/ml IL-
3, 10 ng/ml IL-6, 20 ng/ml G-CSF and 3 units/ml erythropoietin (EPO) (StemCell
Technologies Inc., Vancouver, BC, Canada), at 24 h post-electroporation36. After 2 weeks
of incubation, CFUs were classified and enumerated by trained operators on the basis of
size and morphological characterization under a light microscope.
2.9!Statistics
All statistics analysis were performed by the T-TEST (Excel).
18! !

Chapter 3.!Results
3.1! Examine the potential of heparin binding-deficient AAV6 as liver de-targeting
vector and enhance transduction in HSC
3.1.1! K531E mutant showed reduced heparin binding affinity
To understand the capsid-cell receptor interaction, the sequence of AAV6 and AAV1
capsid protein was compared in the previous studies. The two serotypes have a high degree
of sequence homology (99.2%) but have different affinity to HSPG. AAV6 has moderate
binding to HSPG while AAV1 has weak HSPG binding ability (Halbert, Allen, & Miller,
2001; Z. Wu, Asokan, Grieger, et al., 2006). Six amino acid difference was found between
capsid sequence of the two serotypes. At the threefold conformation, the position of the
K531 residue forms a continuous basic patch similar to AAV2 capsid HSPG binding site
with the surrounding residues at the threefold conformation. It was believed that this basic
patch conformation facilitates electrostatic interaction with the negatively charged sulfate
and carboxyl groups of HSPG. Mutate K531 to Glutamic acid reduced the Heparin binding
and liver transduction level of AAV6 to that of the AAV1 (Z. Wu, Asokan, Grieger, et al.,
2006).
To evaluate the effect of heparin binding mutant on HSC, K531E mutated AAV6 capsid
was cloned by mutagenesis PCR. We produced AAV vectors carrying wild type or K531E
mutant AAV6 capsids. The heparin binding affinity of the mutant was then verified by the
heparin affinity column. Same amount of wild type and K531E vectors, as quantified by
19! !
qPCR, was applied to the column separately. The vectors were eluted out by increasing
concentration of NaCl. The amount of vector genome in each elution fraction was then
quantified by qPCR. The results showed that K531E had weaker heparin-binding with the
elusion peak measured at 30 mM NaCl. In contrast, wild-type capsid AAV6 had the elution
peak at 330 mM NaCl (Fig. 1). The results confirm that K531E mutation reduces heparin-
binding ability of AAV6.
3.1.2! K531E mutation reduce transduction in liver cells and HSC
To examine the transduction ability of the mutant, we generated AAV6-WT or AAV6-
K531E vectors carrying the GFP expression cassette driven by CMV promoter. The vector
genome titers were determined by qPCR. Different cell types (HeLa, HepG2, and HSC)
were transduced with WT and the K531E mutant with indicated vgs/cell. HeLa cells were
used as a positive control since it was reported that K531E mutation on AAV6 capsid does
not impact the transduction ability of the mutant in HeLa cells (Xie, Lerch, Meyer, &
Chapman, 2011). HepG2 cell line represents liver cells. As shown in Fig. 2, our results
show that the GFP transgene expression using WT and AAV6-K531E was at the similar
level in HeLa cells. On the other hand, K531E reduced transduction about ten fold in
HepG2. This result verified the liver de-targeting potential of the K531E mutant. However,
the transduction of the mutant also decreased in HSC about three-fold. In summary, using
the heparin binding mutant, we found that AAV6 transduction on HSC might be partially
dependent on HSPG binding. The K531E mutant has decreased transduction efficiency
both in liver cell line and HSC. However, the reduction in HepG2 is higher than in HSC.
These results indicate that K531E might be a suitable vector for in vivo liver de-targeting
purpose. Less transduction in the liver might increase the available vectors in the
20! !
circulatory system. The increasing circulating vectors will potentially increase the
transduction efficiency in other tissues. However, in the cell type like HSC, the beneficial
effect of liver de-targeting may be limited since HSC also potentially require HSPG
binding for transduction.
3.2! Enhance transduction by preventing proteasome degradation during intracellular
trafficking
3.2.1! Double and triple tyrosine mutation has no positive effect on HeLa or HSC
transduction
Inefficient intracellular trafficking is one of the rate-limiting factors in AAV-mediated gene
delivery. Is was discovered that AAV2 phosphorylation on the AAV2 capsid protein by
epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) leads to capsid
ubiqutination and proteasome degradation. This process was proposed to be the major
reason for decreasing vector genome being delivered to the nuclease. Mutations of the
surface-exposed tyrosine resides might allow the vector to evade phosphorylation and
subsequent ubiqutination and prevent from proteasome degradation. Seven of the tyrosine
mutations on AAV2 were found that increase transduction dramatically both in vitro and
in vivo (Zhong et al., 2008). Six of those residues has homology site on AAV6 and three
of them (Y445F, Y705F, Y731F) were previously proved in muscle (Hakim et al., 2014;
Qiao et al., 2010) and HSC (Song, Kauss, et al., 2013; Song, Li, et al., 2013) that can
improve the transduction once mutates the tyrosine into phenylalanine. The double mutant
showed an even higher improvement in HSC when combining with scAAV6.
21! !
To examine the potential of using tyrosine mutant to enhance transduction, AAV6 vectors
carrying double (Y705F+Y731F) or triple (Y445F+Y705F+Y731F) tyrosine mutations in
the capsid were produced. Both mutants carry the CMV-GFP expression cassette for
transduction efficiency analysis. HeLa cells were transduced with WT and tyrosine mutant
with indicated MOI. Cells were harvested and subjected to FACS analysis at 48 hours after
transduction. As shown in figure 3A, both double and triple mutant has similar transduction
efficiency as WT AAV6 in HeLa cells. The transduction efficiency was also examined at
different days post transduction in HeLa cells. From 1 to 5 days after transduction, all the
mutant showed lower transduction when comparing to the WT (fig. 3B). To further
examine whether the mutation has the cell type-specific effect, we also tested HSC
transduction by WT and double tyrosine-mutant AAV6 vectors. The results show that
double tyrosine mutant and AAV6 has similar transduction ability in HSC (Fig. 3C). In
summary, the tyrosine mutations did not enhance AAV6 transduction in our hands. More
details will be discussed in the discussion
3.2.2! Proteasome inhibitor LLnL enhance gene delivery by AAV6 in HeLa cells
An alternative way to inhibit proteasome degradation is using proteasome inhibitor
treatment. Several proteasome inhibitors have been reported that can increase AAV
transduction. However, the enhancement is cell type specific (Duan et al., 2000). LLnL (N-
acetyl-L-leucyl-L-leucyl-leucinal, also called MG101) works as a peptide aldehyde
inhibitor that bind to the active site of proteolytic enzymes within the proteasome core and
reversibly block its function (Rock et al., 1994). It was reported that LLnL can increase
AAV transduction in HeLa (Grieger & Samulski, 2005) and human airway epithelia
(Jennings et al., 2005) and liver (Duan et al., 2000). Moreover, it was known that can
22! !
increase the transgene expression even more when the genome size is over AAV genome
limitation (4.7 kb) (Grieger & Samulski, 2005). So far, no examination regarding the effect
of LLnL on HSC is reported.
To examine whether LLnL can enhance AAV6 transduction, the treatment was tested in
HeLa cells first. HeLa cells were co-treated with AAV6 and LLnL. Cells were harvest at
72 hours after treatment, and the total DNA (genomic and viral) was extracted. Samples
were then subjected to qPCR analysis to quantify the viral DNA accumulation in cells. We
observed that LLnL treatment increased the amount of vector genome per cell about two-
fold (fig. 4). The enhancement of viral DNA accumulation was observed at two different
MOIs. To further examine whether this improvement also increases the transgene
expression, cells were collected at 48 hours after transduction for GFP expression analysis.
Compare to the 0 uM LLnL treatment, 40 uM LLnL increased the GFP expression for 3
fold (fig. 5A). A lipofectamine transfection control was also included in this experiment.
500 ng of ITR-CMV-GFP plasmid was transfected into cells by Lipofectamine treatment.
The transfection was also co-treated with 0 and 40 ul LLnL. The results show that LLnL
did not increase the GFP expression from lipofectamine-transfected plasmids (fig. 5B).
This result demonstrates the hypothesis that proteasome inhibitor enhances the
transduction through affecting AAV transduction but not just affecting at the gene
expression stage.
23! !
3.2.3! Proteasome inhibitor LLnL enhance gene disruption by AAV6 delivered in HeLa
cells
Having shown that LLnL can enhance AAV6-delivered GFP expression, we then tested
whether LLnL can also enhance other transgene expression and function. We generate the
AAV6 with the transgene that express saCas9 targeting CCR5 locus. HeLa cells were
transduced with AAV6-saCas9 in the presence of LLnL. Cells were harvested 72 hours
post transduction and the genomic and viral DNA was extracted. qPCR analysis showed
that LLnL increased saCas9 gene accumulation in cells by up to 8 fold (fig. 6). The greater
magnitude of viral DNA accumulation compared to AAV6-EGFP transduction suggests
that proteasome inhibitor might have a different impact on different vector genomes. Of
note, in the absence of LLnL, the SaCas9 AAV vector genome in cells was two-fold lower
than GFP AAV. However, the LLnL treatment enhanced saCas9 DNA in cells to the same
level of GFP. The enhancement of gene delivery led to more efficient nuclease activity as
the CCR5 gene disruption increased by two fold (fig. 7). In summary, LLnL increases
transgene delivery and function through AAV6 transduction in HeLa cells. For the gene
therapy purpose, the next important question will be whether LLnL can enhance AAV6
transduction in HSC.
3.2.4! Proteasome inhibitor LLnL toxicity on HSC
We tested a range of LLnL concentrations on HSC to determine the optimal dosage that
has an effect on transduction with limited toxicity. Different from cancer cell line, HSC is
very sensitive to drug treatment. The cell viability after LLnL administration was analyzed.
Comparing to 73% survival rate in mock treatment, LLnL concentration up to 5 uM only
24! !
decreased viability to 63%. However, at concentrations higher than 10 uM, viability
decreased to less than 50% (fig. 8). As a result, 0-5 uM LLnL was applied in further
experiments.
3.2.5! Proteasome inhibitor LLnL increase AAV6 delivered GFP expression in HSC
LLnL was co-administrated with AAV6-CMV GFP to analyze its potential to enhance
transgene delivery in HSC. Cells were harvest at 48 hours after treatment. As shown by the
representative FACS plot in figure 8a, without proteasome inhibitor, AAV6 transduction
at the MOI of 104 gave 20% GPF positive cells. Transduction at the MOI 10
5
gave 32%
GFP cells. Interestingly, co-treated with 5 uM LLnL and AAV6 at the MOI of 10
4

significantly increased transduction efficiency up to 32%(fig. 9A, B). In summary, LLnL
increased AAV6 mediated gene delivery in HSC. The results indicated that with LLnL
treatment, there might be potential to reach the same delivery rate with a log lower MOI of
the viral vector.
3.2.6! LLnL administration affect HSC proliferation and differentiation
To further evaluate the potential of using LLnL in HSC gene editing in vivo, the
differentiation and proliferation ability of HSC after treatment was examined. HSC were
co-administrated with LLnL and AAV6-EGFP at 10
3
vgs/cell. Treated cells were seeded
in methylcellulose medium for triplicates 24 hours after transduction. Colony distribution
was calculated after two weeks incubation. The results show no difference between BFU-
E and CFU-GM colony counts after LLnL treatment. However, the most primitive
population, CFU-GEMM has significantly decreased with 5 uM LLnL (p< 0.01, T-TEST).
The total cell count also showed a significant difference between LLnL treated and non-
25! !
treated group (p< 0.05, T-TEST) (fig. 10). In summary, our data suggest that although
LLnL has the potential to increase AAV6 transduction in HSC, it reduced the stemness
properties of the cells. Therefore, this might not be the suitable treatment for increasing
gene transfer for HSC ex vivo gene editing.
26! !
Chapter 4.!Discussion
4.1!The potential of heparin binding-deficient AAV6 as liver de-targeting vector and enhance
transduction in HSC
HSPG is the primary cell receptor for AAV2 infection (Summerford, Samulski, Hill, &
Carolina, 1998). The HSPG binding site on the AAV2 capsid has been extensively studied
(Perabo et al., 2006). A clustering of basic residues, particularly R585 and R588, has been
identified through mutagenesis studies to meidate heparin binding (Opie, Warrington,
Agbandje-McKenna, Zolotukhin, & Muzyczka, 2003). Individual mutations of R484 and R585
to glutamic acid have been shown to abolish liver transduction of in vivo injected AAV2 in
mice (Kern et al., 2003). On the other hand, the cell receptors of AAV6 are identified as sialic
acid (Z. Wu, Miller, Agbandje-McKenna, & Samulski, 2006) and heparan sulfate (Z. Wu,
Asokan, Grieger, et al., 2006). In order to identify heparin-binding residues on the AAV6
capsid, the AAV1 capsid has been used as a basis for comparison. There are only six amino
acid difference between AAV1 and AAV6 capsids. However, in contrast to AAV6, AAV1
only binds to sialic acid (Z. Wu, Miller, et al., 2006) and has low affinity to heparin (Z. Wu,
Asokan, Grieger, et al., 2006).The role of each of the six amino acids in heparin binding has
been examined in a structure-function analysis of receptor-binding in AAV6. One (Phe129) is
present in the VP1 region in only 13% of subunits. Two of them (Asp418, His642) are far-
removed from the receptor binding and located on the inner surface of the capsid. Only three
(Lys 531, Ler584 and Val598) are surface exposed (Xie et al., 2011). Site-specific mutation at
the six sites in AAV6 and AAV1 revealed that only Lys531 impacted heparin binding (Z. Wu,
Asokan, Grieger, et al., 2006). Transduction in liver cells with the K531E mutant showed 5
27! !
fold decrease compare to wild type AAV6 capsid but no impact was observed in HeLa cells.
Moreover, portal vein infusion in mice also showed about 5 fold reduction in gene expression
when compare K531E mutant to wild type AAV6 capsid (Z. Wu, Asokan, Grieger, et al.,
2006). Therefore, we set out to investigate the effect of K531E mutation on HSC transduction
by AAV6. Our data supports the potential utility of heparin binding-deficient AAV6 as a liver
de-targeted vector. Mutant vector showed a 10-fold decrease of transduction in HepG2 cells
relative to the wild type.
Although AAV6-K531E shows liver de-targeting, it concurrently decreases transduction of
HSC, raising concerns of its usefulness for in vivo application. In our ex vitro study, the mutant
reduced transduction in HepG2 by 10 fold while reducing the transduction in HSC by 3 fold.
Heparin-binding mutation on AAV2 capsid is reported to abolish liver transduction while
increasing the transgene expression in the heart. This enhancement might be due to reduced
retention by the liver. However, the data showed no change in lung and kidney transduction,
suggesting that heart tissue must be significantly permissive to the mutant to allow increased
transgene expression (Kern et al., 2003). Since HSPG is not internalized upon binding to a
ligand when serving as a receptor, indicating that it is a receptor for attachment but not
infection, the transduction enhancement of capsid mutant is potentially due to either the
mutation facilitating the interaction with secondary receptors, or the new capsid conformation
facilitating post-entry processes such as capsid uncoating. However, our in vitro data showed
that heparin binding also plays a role in AAV6 transduction of HSC, indicating that the K531E
mutation might also reduce transduction of HSC in vivo. To achieve the goal of increasing
HSC transduction in vivo, other strategies to increase HSC targeting while achieving liver de-
targeting will likely be required. A recent study reported off-target gene delivery by DARPIN
28! !
modified AAV viral vector. The data showed strong CD4 cell re-targeting through combining
heparan sulfate binding mutation and a CD4 DARPIN modification. The drawback of this
strategy is the difficulty of CD4 DARPIN modified AAV production. However, other
modification specific for HSC might offer a potential strategy for re-targeting AAV6 to HSC
in vivo.
Despite decreased HSC transduction, which potentially limits its clinical usefulness, AAV6-
K531E nonetheless reveals insights into the receptor usage by AAV6 in HSC. K531E mutant
does not abolish liver transduction completely, implying different receptor requirement
between AAV2 and AAV6. HSPG is a primary receptor required for AAV2 infection.
Competition assay using soluble heparin inhibit AAV2 transduction both in HepG2 and HeLa
cells. However, the heparin binding mutation of AAV6 did not decrease transduction in HeLa
cells (Z. Wu, Asokan, Grieger, et al., 2006) indicating that HSPG is not a required receptor for
AAV6 transduction in all cell types. As to date, the receptor usage in HSC for AAV6 is still
unkown. Further studies using heparinase or sialic acid to block different glycosaminoglycan
receptors is required to better understand the interaction between HSC and AAV6.
4.2!Tyrosine mutation on AAV6 capsid has no positive effect on HeLa or HSC transduction
In our results, we demonstrated that double and triple tyrosine mutation did not enhance the
transduction efficiency in HeLa and HSC. This might be due to the difference impact of the
mutation on ss and scAAV6. Seven single tyrosine mutations on AAV2 capsid were shown to
increase transduction in HeLa in vitro and hepatocytes in vivo. It was demonstrated that the
increased transduction efficiency of mutant vectors is due to a lack of ubiquitination and
improved intracellular trafficking to the nucleus (Zhong et al., 2008). Further study
29! !
demonstrated that combined double or triple tyrosine mutations increase the transduction more
than single mutations (Markusic et al., 2010). However, the enhancement on scAAV2 is about
two-fold higher than ssAAV2. This indicates the tyrosine mutant has different impact on ss
and scAAV2. Although it has been reported that scAAV has higher transduction rate compare
to ssAAV (Douglas M McCarty, 2008), scAAV only carry half of the ssAAV genome size (4.7
kb). This limits the application of scAAV in genome-editing studies since our target nuclease
for gene editing, saCas9, has the size of 4.8 kb.
Moreover, although tyrosine mutations were shown to increase ss and scAAV6 transduction
in HSC (Song, Kauss, et al., 2013; Song, Li, et al., 2013) it is unclear if the enhancement is
due to decreased ubiquitination. Further molecular level experiments are needed to verify that
the mutation reduces the phosphorylation and ubiquitination on AAV6.
4.3!Proteasome inhibitor LLnL increase gene transfer and disruption by AAV6 gene delivery
in HeLa and HSC
Our data support previous reports that proteasome inhibition increases nuclear AAV genome
accumulation and leads to higher expression of GFP or gene disruption through saCas9 cutting.
Interestingly, when comparing the intracellular DNA accumulation of AAV6-delivered GFP
and saCas9, saCas9 DNA was two-fold lower than GFP DNA in HeLa cells. LLnL increased
GFP DNA (2781 bp) for 2 fold and increase saCas9 (4851 bp) DNA for up to 8 fold. The effect
of LLnL was dependent on the AAV6 genome size. LLnL increased the saCas9 DNA
accumulation to the same level as GFP. In accordance with our results, it was reported that
AAV encapsidating larger genomes has lower infectivity. Moreover, the co-administration of
proteasome inhibitor was shown to augment transduction of vectors carrying larger genomes
30! !
(6019 bp) to the level similar to those carrying smaller genomes (4675 bp) (Grieger &
Samulski, 2005) This is indicating that AAV vectors with larger genome (bigger than wild type
genome size 4.7 kb) are more susceptible to proteasome degradation and leads to lower
transduction efficiency. The finding that proteasome inhibitor improves larger genome vector
is exciting due to the packaging limitation of AAV, which has been one of the standing
challenges of using AAV as gene therapy vector.
In HSC system, we also showed that LLnL increased gene delivery through AAV6-GFP
transduction. However, so far we have been unable to achieve AAV6-delivered saCas9 activity
in HSC. This might due the combination of higher MOI required for HSC transduction and the
lower transduction efficacy for the AAV has the genome size larger than 4.7 kb. Although we
did not observe gene disruption, I will assess the level of saCas9 DNA accumulation in HSC
to examine whether it is increased by LLnL treatment. Higher titer of viral transduction might
be needed to achieve gene disruption in HSC.
Use of proteasome inhibitors in HSC also warrants careful evaluation of toxicity in primitive
stem cells. In our CFU examination, LLnL decreased total colony count and CFU-GEMM
colonies significantly. Indicating the negative impact of LLnL treatment on HSC stem cell
function. Santoni de Sio et al reported that proteasome inhibitor MG132 treatment strongly
enhanced gene transfer by the lentiviral vector in HSC. Interestingly, no detrimental effect of
MG132 on the stem cell repopulating ability was observed. The engraftment level with or
without drug treatment was similar (Santoni De Sio, Cascio, Zingale, Gasparini, & Naldini,
2006). In addition to MG132, bortezomib (PS-341) is another proteasome inhibitor being
evaluated in a randomized trial in patients with multiple myeloma, a disease frequently treated
with autologous stem cell transplantation. The effect of bortezomib treatment on hematopoetic
31! !
function was assessed through colony growth and engraftment in a murine bone marrow
transplantation model. The results showed no difference in CFU formation and egraftment
when comparing bortezomib and saline treated HSC, indicating that bortezomib treatment does
not compromise HSC function (Koreth, Alyea, Murphy, & Welniak, 2009). Different
proteasome inhibitors are known to act through different mechanisms (Mitchell & Samulski,
2013). The different mechanisms might result in different levels of toxicity in HSC. In our
study, we showed that LLnL can increase transgene expression in HSC but also had a negative
impact on HSC differentiation. Transplantation experiments in a humanized mouse model are
needed to verify the impact of LLnL on primitive HSC. If LLnL results in excessive toxicity
in HSC, we can test alternative proteasome inhibitors such as MG132 and bortezomib for their
activity in enhancing AAV transduction. Both MG132 and bortezomib have been reported to
increase AAV transduction in cell lines. MG132 was reported to increase AAV-mediated
transgene expression in human synoviocytes in vitro and in vivo (Jennings et al., 2005) and
also in human airway epithelia (Yan et al., 2004), while bortezomib was reported to increase
transduction in HeLa cells (Mitchell & Samulski, 2013). Furthermore, bortezomib has
demonstrated efficacy in enhancing AAV transduction when in vivo administered into mice.
Doxorubicin and aclarubicin are two inhibitors that used for cancer treatment also tested has
positive effect on AAV transduction (Yan et al., 2004). With the goal of modifying HSC
without affecting engraftment ability these drugs should also be tested for their impact on AAV
transduction of HSC.In HSC system, we also proved that LLnL increased gene delivery
through AAV-GFP transduction. However, so far we have been unable to achieve AAV6-
delivered saCas9 activity in HSC. This might due the combination of higher MOI required for
HSC transduction and the lower transduction efficacy for the AAV has the genome size larger
32! !
than 4.7 kb. Although we can not see the gene disruption effect. The DNA accumulation data
in HSC is needed to verify that LLnL has increased saCas9 DNA delivery. And higher titer of
viral transduction might be needed to observed the gene disruption in HSC.
Use of proteasome inhibitors in HSC also warrants careful evaluation of toxicity on primitive
stem cells. In our CFU examination, LLnL decreased BFU-E colonies significantly but has no
significant effect on CFU-GEMM colonies or total colony counts assumption, data under
calculation). Santoni de Sio and the group reported in 2006 that proteasome inhibitor MG132
treatment strongly enhanced gene transfer by the lenti-viral vector in CD34+ cells.
Interestingly, no detrimental effect of MG132 on the stem cell repopulating ability was
observed. The engraftment level with or without drug treatment is similar (Santoni De Sio et
al., 2006). Bortezomib (PS-341) is a novel proteasome inhibitor being evaluated in a
randomized trial in patients with multiple myeloma, a disease frequently treated with
autologous stem cell transplantation. The effect of bortezomib treatment on Hematopoetic
function is assessed through colony groith and engraftment in a murine bone marrow
transplantation model. The results showed no difference in CFU formation and egraftment
when comparing bortezomib and salin treated HSC. Indicating that bortezomib treatment does
not compromise HSC function (Koreth et al., 2009). It is known that different proteasome
inhibitor inhibits proteasome activity through different mechanism (Mitchell & Samulski,
2013). This might reflect on the different level of toxicity in HSC administration. In our study,
we prove that LLnL can increase transgene expression in HSC but it also has impact on the
HSC differentiation. Further engraftment experiments are needed to verify the impact of LLnL
on primitive HSC. Other than LLnL, several proteasome inhibitors have also been reported
that can increase AAV transduction in cell lines. MG132 was reported that can increase AAV-
33! !
mediated transgene expression in human synoviocytes in vitro and in vivo (Jennings et al.,
2005) and also in human airway epithelia (Yan et al., 2004). Bortezomib is a FDA-approved
and clinically used drug that known can increase transduction in HeLa cells and in mice
(Mitchell & Samulski, 2013). Doxorubicin and aclarubicin are two inhibitors that used for
cancer treatment also tested has positive effect on AAV transduction (Yan et al., 2004). With
the goal of modifying HSC without affecting engraftment ability these drugs should also be
tested for their impact on AAV transduction of HSC.
34! !
Figures

Figure 3. Heparin elution profiles for AAV6 and K531E mutant.
5x10
8
vg of the vector was applied to a heparin affinity column. Fractions from different salt
concentrations of elution were collected. The vector genome in each fraction was examined by
qPCR.
0.0E+00
1.0E+08
2.0E+08
3.0E+08
4.0E+08
5.0E+08
30 (wash)
130
230
330
430
530
630
730
830
930
1030
Total vector genome
mM NaCl
AAV6
AAV6-K531E
35! !

Figure 4. Transduction comparison of AAV6 and K531E mutant in HeLa, HepG2 and HSC.
HeLa cells were transduced with the MOI of 1000. HepG2 and HSC were transduced with the
MOI of 10,000. Cells were harvest at 48 hours after transduction. The percentage of GFP-
expressing cells were analyzed by FACS. The transduction rate of the AAV6 in each cell type
was considered as 100% efficiency. GFP % from K531E transduction were normalized by AAV6
transduction rate and shown as reduction rate.
0
20
40
60
80
100
120
HeLa HepG2 HSC
K531E transduction reduction compare
to AAV6 (%)
AAV6
K531E
36! !
(A)!

(B)!

(C)!

0
20
40
60
80
100
120
100 1000 10000 100000
GFP %
MOI
AAV6
AAV6_Y705F+Y731F
AAV6_Y445F+Y705F+Y731F
0
5
10
15
20
25
30
1 2 3 4 5
GFP %
Days post transduction
AAV6
AAV6_Y705F+Y731F
AAV6_Y445F+Y705F+Y731F
0
10
20
30
40
10000 100000
GFP (%)
MOI
AAV6
AAV6-
(Y705F+Y731F)
37! !
Figure 5. tyrosine mutant transduction in HeLa and HSC.
(A)! Transduction efficiency of AAV6 and tyrosine mutant in HeLa cells. MOI from 100 to
100,000 were tested. The transduction efficiency showed dosage dependent, but the
tyrosine mutation has no positive effect in all MOI treatment. The standard deviation
from two independent experiment were shown.
(B)! Transduction efficiency of WT AAV6 and tyrosine mutant in HeLa cells at different
days post transduction. Cells were transduced at 1000 MOI.
(C)! Transduction efficiency in HSC. MOI of 10,000 and 100,000 was applied in the
experiment. The tyrosine mutation showed no positive effect in both MOI transduction.

38! !
(A)

(B)

Figure 6. Proteasome inhibitor increased viral DNA accumulation in HeLa cells transduced with
AAV6-GFP.
Cells were transduced with MOI of 1000 (A) and 100,000 (B). The vector genome copy number
was detected by qPCR targeting ITR. The results were normalized by RNaseP copy number. The
amount of vector genome per cell with or without LLnL treatment were calculated.

0.0
20.0
40.0
60.0
80.0
100.0
120.0
140.0
Mock AAV6-EGFP AAV6-EGFP +
LLnL 40 uM
Viral DAN accumulation in cells
(vg/cell)
1000 MOI
0
2000
4000
6000
8000
10000
12000
Mock AAV6-EGFP AAV6-EGFP +
LLnL 40 uM
Viral DAN accumulation in cells
(vg/cell)
100000 MOI
39! !
(A)

(B)

Figure 7. LLnL increased GFP expression with AAV6 transduction in HeLa cells.
(A) HeLa cells were transduced with AAV6-GFP at the MOI of 1000. LLnL was co-administrate
at the same time of transduction. The GFP % was analyzed by FACS at 48 after transduction. The
results are from two independent experiments.
(B) ITR-CMV-GFP plasmid was transfected into HeLa cells by Lipofectamine treatment. The
LLnL was co-administrate at the same time of transduction. GFP-positive cells were analyzed by
FACS at 48 hours after transduction.
0
2
4
6
8
10
12
14
16
18
0 uM 40 uM
GFP (%)
LLnL
0
10
20
30
40
50
60
70
80
90
100
0 uM 40 uM
GFP (%)
LLnL
40! !
(A)

(B)

Figure 8. Proteasome inhibitor increased viral DNA accumulation in HeLa cells transduced with
AAV6-SaCas9.
Cells were transduced with MOI of 1000 (A) and 100,000 (B). The vector genome copy number
was detected by qPCR targeting ITR. The results were normalized by RNaseP copy number. The
amount of vector genome per cell with or without LLnL treatment were calculated.

0
20
40
60
80
100
120
Mock sAAV6-saCas9 sAAV6-saCas9 +
40 uM LLnL
Viral DAN accumulation in cells
(vg/cell)
1000 MOI
0
1000
2000
3000
4000
5000
6000
7000
Mock sAAV6-saCas9 sAAV6-saCas9 +
40 uM LLnL
Viral DAN accumulation in cells
(vg/cell)
100,000 MOI
41! !

Figure 9. LLnL increased gene disruption by AAV6 delivered CCR5 targeted SaCas9 in HeLa
cells.
Results are from three independent experiment. Two different home made AAV-SaCas9 prep were
applied. Indicating the reproducibility of targeted nuclease AAV production in our hand.

0
2
4
6
8
10
12
14
16
18
20
0 uM 20 uM 40 uM
CCR5 Gene disruption ( %)
LLnL
42! !

Figure 10. Cell toxicity analysis of LLnL treatment on HSC.
HSC were treated with increasing concentration of LLnL from 0 uM to 20 uM. Cells were harvest
at 24 hours after treatment and stained for 7AAD and Annexin V. The viability rate indicating the
healthy cells showed 7AAD
-
and Annexin V
-
.
0
10
20
30
40
50
60
70
80
0 uM 2.5 uM 5 uM 10 uM 20 uM
Survival (%)
LLnL
43! !
(A)

(B)

Figure 11. LLnL increased AAV6 delivered GFP expression in HSC.
(A)! Representative data from flow cytometry showed GFP % in different treatment condition.
(B)! GFP expression increased with 5 uM LLnL treatment. The results are from three independent
experiment (three different HSC donor). Compared to 0 uM LLnL, student T-test showed
significant enhancement with 5 uM LLnL treatment. (p<0.05)
0
5
10
15
20
25
30
35
0 uM 2.5 uM 5 uM
GFP (%)
LLnL
* (p<0.05)
44! !
(A)

(B)

Figure 12. LLnL administration affect HSC proliferation and differentiation.
HSC were subjected to colony-forming unit assay at 24 hours post transduction/LLnL
administration. Standard deviation from triplicate sample total colony count are shown. Student t-
test showed significant difference of total colony count (p<0.05).

0
20
40
60
80
100
120
140
160
180
0 uM 5 uM
LLnL
Total colony count
0
20
40
60
80
100
120
0 uM 5 uM
LLnL
Colony distribution (%)
CFU-GEMM
CFU-GM
BFU-E
*(p<0.05)
* (p<0.05)
45! !
Appendix

Modified AAV production protocol for Cannon Lab
*The red check points in the protocol indicate the quality control for each step. Please make sure your prep
match to each check point before you proceed to the next step.
1.! Preparation of 293 AAV cells for transfection (1 week before transfection)
a.! D1: Thaw two tubes of 293 AAV (P6, in AAV production stock box) in a 125T
flask
b.! D3: split the cells into 4 125T flask
c.! D6: seed the cells in ten 15ml dish (9x10
6
/ dish) and proceed to the transfection the
next day (one confluent 125T would have around 3x10
7
cells)

46! !
2.! Transfection of viral plasmid
a.! 24 hours before transfection, plate the cells in the 15 cm dish, 9x10
6
cells/ dish (in
20ml medium). Prepare 5 dishes for a vector
b.! If your vector doesn’t express GFP, prepare at least an extra plate for AAV6-cGFP
transfection as control
c.! The next day, replace fresh medium before transfection
d.! Prepare the transfection mixture
i.! Calculate the required amount of plasmid follow the table below
ii.! Mix water, plasmid and 2M CaCl2 in a 15 ml tube, mix well by vortex

per 15 cm dish
pITR- GFP 27 ug
pHelper 27 ug
pRC6 27 ug
CaCl
2
2 M
water Up to 1350

e.! Drop wise add the mixture from [d] (1350 ul/dish) 2x HBS in 50 ml tube
i.! Add 2xHBS in the 50 ml tube first (thaw the HBS at RT)
ii.! Set the tube at an angle to increase the surface area of the liquid
iii.! Add the mixture from [d] drop by drop into the 2xHBS, the drops should
equaly distributed on the liquid surface
iv.! The pH of the 2xHBS should be 7.12~7.14, this is a critical range, lower or
higher pH will results in bad transfection efficiency
f.! Wait for 5 min (very critical, do not wait longer), you should see white particles
form in the mixture
g.! Before adding the mixture into cells, mix it well by pipet up and down
h.! Add the mixture dropwise to the dish, evenly distribute on the plate and prevent
suspend the cells
47! !
i.! After this step, look under microscope, you should see the particles like
small dust well distributed over the plate. If the particles form cluster, it will
decrease the transfection efficiency and increase the cell death
i.! 16 hours after transfection, wash with PBS once
j.! Add 20 ml fresh 10% FBS DMEM
k.! Check the GFP 24h after transfection, you should have 90% GFP cells
i.! If the GFP% less than 50, the transfection efficiency is too low, do not
proceed. Go back to the previous steps: check the pH level of HBS or other
parameter.
Check point: the CMV-GFP positive cells should reach 80%

Bright!field
48! !
3.! Harvesting of rAAVs
a.! 72 hours after transfection, remove media from cell culture plates.
b.! Gently wash the cells in warm PBS
c.! Add 10 ml PBS to one plate and gently remove cells with a cell scraper
d.! Transfer the pellet containing PBS to the next plate and add 10 ml fresh PBS to the
first plate to wash down the remaining cells
e.! Scrap down the cells from the second plate, transfer the PBS to the third plate, and
use the PBS from [d] to wash down the remaining cells on second plate
f.! Repeat c-e until harvest all the plate, collect all the PBS in a 50 ml tube
g.! Wash the plate with 10 ml (or more if needed) PBS once and collect the PBS
h.! Fill the tube to total 50 ml PBS
i.! Pellet cells at 800 x g for 10 minutes.
j.! Discard supernatant, freeze the pellet in -80°C
i.! If your vector express GFP, the pellet should be green
Check point: If your vector expresses CMV-GFP, the pellet should be green

4.! Lysing of cells
a.! Thaw the cell pellet
b.! For 5 dishes transfection, prepare 10 ml (2ml/ dish)150Mm NaCl, 20mM Tris
pH8.0
c.! add 0.5% sodium deoxycholate in [a]
d.! Add 100 unit/ml benzonase (250 unit/ul), or DNase with the same total units
e.! Re-suspend cell pellet
f.! Incubate in 37 °C, 2 hr
g.! Centrifuging at 3000g for 15 mins
h.! Transfer the supernatant to a new 50 ml tube
i.! Filter by 0.22 uM filter into 50 ml tube
j.! Sample can be stored at -80°C before continuing
k.! Proceed to heparin column purification or iodoxinal gradient purification

49! !
5.! Heparin column purification of rAAVs (2 - 3 hours) (McClure, Cole, Wulff, Klugmann,
& Murray, 2011)
a.! Setup HiTrap heparin columns using a peristaltic pump so that solutions flow
through the column at 1 ml per minute for steps 5.2 to 5.4. It is important to ensure
no air bubbles are introduced into the heparin column.
b.! Equilibrate the column with 10 ml 150 mM NaCl, 20 mM Tris, pH 8.0.
c.! Apply 50 ml virus solution to column and allow to flow through.
d.! Wash column with 20 ml 100 mM NaCl, 20 mM Tris, pH 8.0.
e.! Using a 5 ml syringe continue to wash the column with 1 ml 200 mM NaCl, 20 mM
Tris, pH 8.0, followed by 1 ml 300 mM NaCl, 20 mM Tris, pH 8.0. Discard the
flow-through.
f.! Using 5 ml syringes and gentle pressure elute the virus from the column by
applying:
i.! 1.5 ml 400 mM NaCl, 20 mM Tris, pH 8.0
ii.! 3.0 ml 450 mM NaCl, 20 mM Tris, pH 8.0
iii.! 1.5 ml 500 mM NaCl, 20 mM Tris, pH 8.0
iv.! Collect these in a 15 ml centrifuge tube.
g.! Wash the column with 10 ml 1M NaCl
h.! Store the column in 20% ethanol

6.! Concentration for column purification (McClure et al., 2011)
a.! Concentrate vector using Amicon ultra-4 centrifugal filter units with a 100,000
molecular weight cutoff.
b.! Load 4 ml of column eluate into the concentrator and centrifuge at 2000 x g for 2
minutes (at room temperature).
c.! Discard flowthrough and reload concentrator with remaining virus solution and
repeat centrifugation.
i.! The concentrated volume should be approximately 250 µl. If concentrated
volume is significantly more than this, discard the flow through and
continue to centrifuge in one minute steps until volume is approximately
250 µl.
d.! Remove the virus from the concentrator, wash the filter with 100 ul PBS, add the
PBS together with virus
e.! Mix well and aliquot the virus 20ul / tube
f.! Proceed to DNA extraction for qPCR titration or GFP titration on HeLa cells

50! !
7.! Iodoxinal gradient purification (from Dan Stone 10/2/13)
a.! Prepare the gradient solutions follow the table
% Iodix 60% 2M NaCl 10x PBS-MK H
2
O ul Phenol
Red
Total
volume
15 50 100 20 30 0 200
25 62.5 15 72.5 225 150
40 80 12 28 0 120
54 108.0 12 0 180 120

b.! Thaw the cell lysate
c.! Pre-chill the rotor (Rotor type: VT65.1, Serial No. 201)
d.! Set up the gradient in 12 ml Ultracrimp tube in the following order
i.! 15%, 1.7 ml
ii.! 25%, 1.7 ml
iii.! 40%, 2 ml
iv.! 54%, 1ml
v.! Lysate, up to 4 ml (from two plates)
vi.! Fill the rest of the volume with PBS (need about 2 ml PBS/tube)
e.! Seal the tube
f.! Proceed to centrifugation
i.! BeckMAN L8-80 Ultracentrifuge (4F)
ii.! RPM 59K
iii.! Time: 1hr and 10 mins
iv.! Turn on vacuum, select accelerate speed at 5 and decelerate speed at 7
v.! Press “auto run”
g.! After the centrifuging, collect the tube back carefully, prevent disturbing the
gradient
h.! The AAV should contain in 40% layer
i.! Use a needle to puncture the first hole on the top of the tube to allow the airflow
j.! Use the same needle, connect to 5ml syringe, puncture the tube at 40%:54% with
the bevel up
k.! Start to harvest the 40% layer
l.! When it gets close to the protein band, turn the bevel down and collect until
approximately 2-3 mm from the band (the band contains cells proteins that are
contaminants, avoid collecting it)
m.! Proceed to concentration for gradient purification

51! !
8.! Concentration for gradient purification
a.! Concentrate vector using Amicon ultra-4 centrifugal filter units with a 100,000
molecular weight cutoff.
b.! Dilute the virus containing 40% layer by PBS up to 15 ml
c.! Load 4 ml of diluted layer into the concentrator and centrifuge at 2000 x g for 2
minutes (at room temperature).
d.! Discard flowthrough and reload concentrator with remaining virus solution and
repeat centrifugation.
i.! The concentrated volume should be approximately 250 µl. If concentrated
volume is significantly more than this, discard the flow through and
continue to centrifuge in one minute steps until volume is approximately
250 µl.
e.! Remove the virus from the concentrator, wash the filter with 100 ul PBS, add the
PBS together with virus
f.! Mix well and aliquot the virus 20ul / tube
g.! Proceed to DNA extraction for qPCR titration or GFP titration on HeLa cells

9.! DNA extraction for qPCR titration

a.! Prepare the mixture
1 prep _____ prep
NEB buf. 3 5
DNase (40U/ul) 1 (1u/ul)
Sample 5
Water 39
total 50

b.! Incubate at 37 °C for 30 min
c.! Inactivate DNase in 75 °C, 10min
d.! Add 50 ul of 2x protease K buf. , 5 ul protease K (20mg/ml)
e.! Incubate at 37 °C for 1 hr
f.! Heat inactivate the protease K at 95 °C for 20 mins

52! !
10.!qPCR titration
a.! Reagents
i.! AAV reference standard material ATCC-1616
ii.! TaqMan® Universal Master Mix II, with UNG Invitrogen
iii.! Primer AAV ITR-Forward 5’-GAACCCCTAGTGATGGAGTT-3’
iv.! Primer AAV ITR-Reverse 5’-CGGCCTCAGTGAGCGA-3’
v.! Probe AAV ITR-Probe 5’-FAM-
CACTCCCTCTCTGCGCGCTCG-Tamra-3’

b.! set the table for qPCR plate (example in Appendix table 1 )
c.! prepare the mixture

mixture
For 1
reaction

TagMan Universal Master Mix 12.5
sample 5
preimer F (100nM) 2.5 of 1uM stock
preimer R (340 Nm) 2.5 of 3.4uM stock
probe (100nM) 2.5 of 1uM stock
PCR cycle
95
o
C 15 min
40 cycles
95
o
C 1 min
60
o
C 1 min

Check point: the qPCR titer should be around 4.7x10
11
(vg/ml). The total yield should be
around 1.14x10
10
(vg/ 15 cm dish)

53! !
11.!GFP titration on HeLa cells
a.! The day before transduction, seed HeLa cells into a 24-well plate at 4E4 cells/well
b.! Right before titration, trypsinize 8 wells, pool and count to obtain cell number/well
c.! Dilute AAV prep as follows in serum-free DMEM:
1st dilution

1:10

(20ul + 180ul media)

1:100

1:250

1:500

1:1000

AA V6 1:10

100

40

20

10

uL

Media

900

960

980

990

uL

1:2000

1:5000

1:10000

AA V6 1:100

20

8

4

uL

Media

980

992

996

uL

d.! Wash HeLa in serum-free DMEM
e.! Add diluted AAV to HeLa, 400ul/well, in duplicate
f.! Incubate overnight; the following day add 400ul/well DMEM 10%FBS
g.! FACS for GFP 48 hours post transduction

Check point: the infectious particle should be around 6.9 x10
-5
% (vg/tu) in HeLa cells (this
is the data from one experiment, needs more repeats and try to scale down in 48 well plate)

12.!References (McClure et al., 2011)

54! !
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Abstract (if available)

Abstract Genome editing in hematopoietic stem cells (HSC) through ex vivo or in vivo approaches are promising strategies for curing several blood cell related diseases such as HIV infection. However, delivery of genome editing reagents has proven to be challenging in HSC in both approaches. ❧ Ex vivo genome editing by Electroporation of ZFN mRNA, which is the current method of choice for ex vivo HSC genome editing, results in ~5% transgene insertion, but the engraftment data reveal that it is still challenging to modify the most primitive HSC. Our previous study using AAV6 as a donor delivery vector successfully achieve high genome editing results (23%) in the most primitive HSC population indicating a promising editing strategy using AAV6. ❧ However, there are two potential disadvantages to the ex vivo approach, First, the isolation and culture of HSC carry some risk of contamination. Second, the ex vivo culture of HSC can reduce their stem cell function. For these reason, an in vivo approach is more desirable. The challenges of using viral vector for in vivo genome editing to date are the low transduction efficiency in vivo, the genome integrating issue and the immunogenicity. Adeno-associated viral (AAV) vector is also an ideal system for in vivo editing purpose, due to their ability to transduce quiescent cells, their transient expression profile upon cell proliferation, and their stability and low immunogenicity in vivo. ❧ Despite all the advantages, there are still difficulties for AAV-mediated delivery of genome editing reagents to HSC. AAV transduction efficiency in HSC is relative low compared to that in cell lines. Furthermore, in vivo HSC transduction efficiency is further lowered due to broad tissue tropism of AAV and the preferential transduction in the liver, causing loss of vectors in targeted tissues. ❧ In this study, we explore potential strategies to enhance in vivo transduction of HSC by AAV vectors by 1) de-targeting AAV vectors from liver cells to increase their availability for HSC transduction in vivo, and 2) improving intracellular trafficking of AAV vectors to increase transgene expression. To promote liver de-targeting, we use AAV vectors carrying capsid proteins with one mutation, K531E, which has been shown to function in AAV binding to liver cells but has an unknown role in binding to HSC. To improve intracellular trafficking and transgene expression, we use capsid tyrosine mutations and proteasome inhibitor treatment to allow AAV vectors to overcome proteasome-mediated degradation. ❧ In the results, we found that the K531E mutant AAV demonstrated a 10-fold decrease in transduction of a liver cell line, with a concomitant 3-fold decrease in transduction of HSC. While these findings support the liver de-targeting activity of the K531E mutant, the decreased HSC transduction limits the usefulness of this AAV for delivery of genome editing reagents to HSC in vivo. In addition, we found that proteasome inhibitor significantly increased AAV transduction in HSC. Whether the functionality of primitive HSC is preserved after proteasome inhibitor treatment remains to be determined.

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Creator Chen, Hsu-Yu (author)

Core Title Improving adeno-associated viral vector for hematopoietic stem cells gene therapy

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 04/21/2016

Defense Date 03/18/2016

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag adeno-associated viral vector,gene therapy,hematopoietic stem cells,OAI-PMH Harvest

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Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Cannon, Paula (committee chair), Gao, Shou-Jiang (committee member), Huang, I-Chueh (committee member)

Creator Email dolphinazuer@gmail.com,hsuyuche@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-238229

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