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INTRODUCTION
Recent advances in the area of gene therapy have given the medical field a new
and powerful approach for finding cures for previously incurable diseases. Gene Therapy
is the insertion, alteration or removal of a single or multiple genes within a cell, or cells,
to treat a disease. This can be accomplished by physical methods of direct DNA transfer
using liposomes, electroporation (methods which are commonly used in the research lab)
and also particle mediated gene transfer such as DNA covered gold micro-particles which
are then moved inside a cell using an electro-mechanical force (“gene gun”). However,
such methods are not always efficient and do not lead to permanent gene transfer,
therefore, these are also called “transient” gene transfer methods (Sheridan 2011).
Viral vectors can also be utilized as gene transfer vehicles and processes already
built into such viruses can be harnessed to make the gene transfer process more efficient
and controllable. These vectors are stripped-down DNA and RNA viruses that penetrate a
target cell and deliver their genetic information into the cell (Sheridan 2011, Varmus et
al. 1997). Examples for DNA viral vectors include Adenoviral vectors or Adeno
Associated Viral (AAV) vectors. Adenoviruses are well known human cold viruses; in
their latest generation, all of their replication competency and pathology causing genes
have been removed and the DNA of interest to be transferred into the target cell can be
packaged into them. In their generation process they can be concentrated by gradient
ultracentrifugation and very high titers, up to 10e14 transducing units per mL, can be
achieved. However, if they are used at high titers for direct injection into a human, they
can still cause inflammation because not all antigenic and inflammatory viral structural
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proteins can be completely removed (Grieger et al. 2005). AAV vectors can solve this
problem since they do not cause pathology in vivo without an adenovirus present as a
helper virus. Again, AAV vectors can be concentrated and purified to very high titers and
are excellent candidates for direct injectable gene therapy vectors due to their in vivo
safety profile. What these DNA vectors are lacking, however, is permanent gene transfer.
AAV vectors will only deliver their genetic payload episomaly, which will be diluted out
with every cell division and will also be degraded over time (Grieger et al. 2005). It
should be pointed out, however, that non-dividing cells such as neurons can harbor
episomal DNA for a much longer period of time. Therefore, AAV vectors are well suited
for direct gene transfer into the human central nervous system (Grieger et al. 2005).
If permanent gene integration into the host genome is required, retroviral vectors
are needed. These vectors were initially based on mouse onco-retroviruses; viruses that
cause leukemia in mice, such as the Moloney leukemia virus (MoLV). These are very
simple viruses that use RNA as the carrier of their genetic information on which specific
regions code for viral structural proteins, viral polymerases which catalyze specific viral
RNA and protein functions, and also the viral envelope which is needed for attachment to
the target cell. Among the viral polymerasesis an important enzyme called “reverse
trahscriptase”, which reverse transcribes the viral RNA into double stranded DNA.
Another viral enzyme called “integrase” then pseudo-randomly integrates the newly
synthesized DNA into the target cell DNA where it remains permanently (Sheridan 2011,
Varmus et al. 1997, Naldini et al. 1996). Viral DNA inside the nucleus is then upregulated to make more viruses by utilizing the cell’s transcription and translation
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machinery – a cycle that continues as long as the cell lives. Such retroviruses can be
made replication incompetent by removing all of their viral genetic information and
replacing it with the RNA of a gene of interest that should be transferred. Utilizing all the
unique viral properties and enzymes, the gene of interest can then be transferred and
permanently integrated into the host cell genome (Varmus et al. 1997, Naldini et al.
1996). This offers the unique opportunity of permanently correcting a gene defect. HIV
belongs to a sub-group of retroviruses called “lentiviruses” which are more complicated
than simple onco-retroviruses because they also carry several regulatory genes that help
with steps in the viral life cycle. However, lentiviruses can also be harnessed as excellent
permanent gene transfer vehicles in the same way as onco-retroviruses since all of the
viral genome can be replaced with a gene of interest that needs to be transferred. It is a
common agreement in the gene therapy community to call onco-retrovirus based vectors
“retroviral vectors” and HIV based vectors “lentiviral vectors”. Interestingly, the size of
the gene of interest that can be packaged into a lentiviral or retroviral vector is about
twice as large as the size of the gene that can be transferred with DNA viruses allowing
for even more complex genes to be packaged. Lentivectors can transduce dividing and
non-dividing cells, while retrovectors are limited to the transduction of dividing cells
because they require the breakdown of the nuclear membrane for DNA integration. Both
types of vector particles can be “pseudotyped”, which means their envelope needed for
attachment of the viral particle to the target cell can be altered and tailored to the specific
cell type that is targeted (Varmus et al. 1997, Naldini et al. 1996).
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Both vectors are produced in “packaging cell lines” which are cell lines that
harbor the genes for the structural proteins, the polymerases and the envelope to make the
viruses, but lack the ability to package these genes into the assembled viral particles. The
gene of interest that needs to be transferred is also present in the packaging cell line and
contains the “packaging signal”, which allows this gene to be packaged into the
assembled vector particle as the payload (Naldini et al. 1996, Anderson et al. 2009). The
finished vector particles are then released from the packaging cell and can be collected
and concentrated. As compared to Adenoviral and AAV vectors, titers achievable with
retro and lentiviral vectors are lower, maximally up to 10e10 transducing units per mL,
but are still greatly sufficient to effectively transducer ex vivo human target cells such as
hematopoietic stem cells, mesenchymal stem cells or fibroblasts. Additionally, all gene
therapy vectors, DNA and RNA vectors, if produced properly, are free of advantageous
viruses, replication competent viruses, bacteria or mycoplasma and endotoxin. This is a
requirement for such vectors being used in human gene therapy applications.
My role in the project described was to develop methods to effectively
manufacture, under Good Manufacturing Practice conditions, novel lentiviral vectors for
ex vivo transductionof cells ultimately used in human gene therapy applications. Good
Manufacturing Practice manufacturing is a requirement given in the United States Code
of Federal Regulations 21, Parts 210 and 211. It specifies that a controlled and highly
clean environment and techniques have to be applied to manufacture pharmaceuticals or
other products that are used in human clinical applications. Additionally, strict quality
control measures have to be applied to the manufacturing facility, manufacturing process
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and the final product. Since very high lentiviral titers are needed for our clinical
applications, a new method of vector concentration utilized by our group required the
development of a Good Manufacturing Practice grade process for this purpose.
Additionally, only clinical grade reagents with certificates of analysis and also cell lines
that were fully certified (absence of advantageous virus and any other contaminants)
could be utilized. It was for me to manufacture the lentiviral vector, under the FDA
mandate that it has to be identical to the one used in human clinical trials, needed to
generate all the Investigational New Drug (IND) enabling studies for a stem cell gene
therapy clinical trial for HIV and also for the treatment of epidermolysis bullosa.
Gene therapy can be an extremely useful technique in the treatment of diseases
because of the ability to correct dysfunctional genes and/or introduce new, beneficial
genes to a genome (Sheridan 2011). Through the use of gene therapy, the researchers at
the UC Davis Institute for Regenerative Cures have explored a novel approach to finding
a cure for HIV infection. Gene therapy has also provided the medical field with maybe its
most powerful discovery ever, the ability to create induced pluripotent stem cells (iPSC)
from fully differentiated somatic cells. Human iPSCs are functionally almost indentical to
human embryonic stem cells and can be differentiated into tissues of all three germ layers
(Tolar et al. 2010). This allows for the creation of patient specific tissues for regenerative
medicine purposes. Patient-specific iPSCs offers new hope for finding a cure for
previously untreatable diseases such as epidermolysis bullosa, a defect in Collagen VII,
which causes skin to blister and dislodge from the underlying connective tissue creating
large wounds. iPSCs can be generated from skin fibroblasts of patients with the disease,
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can be gene corrected for the defect, differentiated into keratinocytes, which in turn can
be expanded and used to produce gene corrected dermal grafts for transplantation onto
the patient to close the wounds with normal skin(Tolar et al. 2010).
The Human Immunodeficiency Virus (HIV) and the subsequent development of
the Acquired Immunodeficiency Syndrome (AIDS) continues to affect millions of people
around the world. There is no effective vaccine available, and current antiretroviral
treatments are only effective at suppressing viral load but do not cure it. The high
mutation rate of HIV has made it difficult to design drugs against it, however, a new stem
cell based gene therapy approach may provide a better alternative for HIV treatment that
may ultimately lead to a functional cure of the disease (Anderson et al. 2009, Winters et
al. 2000, Marks et al. 2004, Martinez-Picado et al. 2000, Lafeuillade et al. 2001,
Bacheler et al. 2000).
HIV infects the CD4+ cells of the human immune system by binding to the CD4
receptor and also the CCR5 co-receptor (macrophage tropic HIV strains) or the CXCR4
co-receptor (T cell tropic HIV strains) on the host cell through interactions with
glycoproteins on the HIV envelope surface (Figure 1). This binding allows the HIV
particle to fuse with the host cell membrane and enter the cytoplasm of the host cell.
Once inside the host cell, the HIV genetic information then uncoats from its core
releasing its “pre-integration complex” into the host cell. Among other components, the
HIV pre-integration complex includes the complete viral RNA genome and the unique
viral enzymes reverse transcriptase and integrase. Reverse transcriptase converts the
RNA genome into double-stranded DNA. The viral capsid, which is also part of the pre-
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Figure 1. HIV Life Cycle. HIV particle attaches to target cell through envelope glycoproteins,
engages CD4 and CCR5 or CXCR4 receptors, fuses with target cell. 2: Reverse transcription
of viral RNA into double stranded viral DNA utilizing viral reverse transcriptase. 3: Transport
of viral DNA into the nucleus through coupling to cellular transport mechanism. 4: Integration
of viral DNA into the host DNA via viral integrase. 5: Transcription of integrated viral DNA
into short and long viral RNAs, transport of RNAs into cytoplasm, translation of RNAs into
viral proteins that are cut to length using viral protease. 6: Assembly of viral particle,
packaging of two viral RNA copies into particle and subsequent budding of the fully
assembled particle from host cell.
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integration complex, then attaches to a cellular transport mechanism and actively
transports the newly synthesized viral DNA into the nucleus. The viral integrase then
pseudo-randomly integrates the double stranded, full length viral DNA into the genome
of the target cell (Figure 1) (Anderson et al. 2009, Winters et al. 2000, Marks et al. 2004,
Martinez-Picado et al. 2000, Lafeuillade et al. 2001, Bacheler et al. 2000).
HIV immediately becomes active after integration due to the action of several
regulatory factors encoded by the virus and initiates transcription of spliced viral RNA.
These short viral RNAs are translated into viral regulatory proteins that induce an
environment in the host cell suited for up-regulation of viral replication and viral particle
synthesis. Infected cells start to produce a large amount of virus (Figure 2), which can
only be kept under control by a vigorous immune response mounted by the host immune
system. This correlates with a huge viral load measurable in the peripheral blood of
newly infected individuals. Only after the immune system has been able to eliminate
most of the circulating virus and particularly the infected cells responsible for high viral
output will a “steady state” be achieved, that determines the set point of the viral load in
the patient. A very long lived, virally infected “reservoir” of target cells will also be
created at that time. This is achieved by viral integration into cells with low cell cycle
turnover rates (Anderson et al. 2009, Winters et al. 2000, Marks et al. 2004, MartinezPicado et al. 2000, Lafeuillade et al. 2001, Bacheler et al. 2000).
In the past, a so called “latent” state of viral infection was proposed. However,
this is not the case in most infected individuals, but is rather an effect caused by the
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Figure 2. Electron Microscope Image of Budding HIV.
Upon infection, HIV is known to immediately up-regulate
expression of viral RNA and to create a burst of virus. This
burst of viral particles budding from an HIV infected cell is
shown here. HIV can be recognized as a small, round
particle with a dark core, which contains viral RNA.
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immune system trying to keep HIV replication at a low level. This state usually
lasts for 8-10 years in untreated HIV infected individuals. However, after HIV has
destroyed the T-cell clones that are responsible for mounting this prolonged immune
response or “latent state”, HIV replication increases dramatically which, destroys the
remaining T cells and leads to the clinical manifestations of AIDS which are severe
opportunistic infections that eventually cause death (Anderson et al. 2009, Winters et al.
2000, Marks et al. 2004, Martinez-Picado et al. 2000, Lafeuillade et al. 2001, Bacheler et
al. 2000).
The current treatment for HIV, highly-active antiretroviral therapy (HAART),
consists of a combined use of multiple drugs that target different stages of the retrovirus
life cycle thus presenting multiple obstacles for viral replication. Only if multiple stages
in the life cycle of the virus are targeted, replication of HIV can be almost completely
shut down, which then limits the amount of mutations HIV can acquire that could
possibly escape drug therapy (Anderson et al. 2009, Winters et al. 2000, Marks et al.
2004, Martinez-Picado et al. 2000, Lafeuillade et al. 2001, Bacheler et al. 2000).
Common classifications of drugs used in HAART are: 1) nucleoside and
nucleotide reverse transcriptase inhibitors (NRTI) which inhibit reverse transcriptase by
being directly incorporated into the newly synthesized viral DNA strand and inhibiting its
elongation, 2) Non-nucleoside reverse transcriptase inhibitors (NNRTI) which inhibit
reverse transcription in a steric manner by binding to the active part of reverse
transcriptase and inhibiting primer binding, 3) protease inhibitors which inhibit the
formation of functional viral proteins by inhibiting the function of the viral protease
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enzyme, which is a unique viral protein synthesized by the virus post integration and
needed to cleave the newly synthesized long protein strands, 4) integrase inhibitors which
inhibit the integration of the viral genome into the host genome by sterically inhibiting
the function of the viral integrase enzyme and 5) entry inhibitors which interfere with
binding, fusion and entry of the viral particle into the host cell (Anderson et al. 2009,
Winters et al. 2000, Marks et al. 2004, Martinez-Picado et al. 2000, Lafeuillade et al.
2001, Bacheler et al. 2000).
HAART has been shown to successfully suppress viral replication if used
properly. However, due to the established viral reservoir, HAART only suppresses viral
replication and does not cure patients of the virus, since the viral reservoir cannot be
eradicated. Patients must therefore be on HAART for the duration of their lives.
Compounding that problem further is the fact that HAART may become toxic to an
increasing number of patients, particularly when they age, as well as produce drugresistant mutants of the virus after prolonged use of the treatment, as there is still some
residual viral replication in some sanctuary sites. Therefore, it is necessary to find
alternative treatments that could circumvent the shortcomings of HAART. Fortunately,
the field of gene therapy provides a promising alternative to the current treatment
methods, and the application of stem cell based anti-HIV gene therapy could provide
patients with a one-time treatment that would confer a lifetime of HIV resistance
(Anderson et al. 2009, Winters et al. 2000, Marks et al. 2004, Martinez-Picado et al.
2000, Lafeuillade et al. 2001, Bacheler et al. 2000).
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Gene therapy for the treatment of HIV is the process of introducing a gene or genes into
the genome of HIV target cells that, when converted into their corresponding gene
products, will provide anti-HIV activity, and protect HIV target cells permanently from
HIV infection and replication (Anderson et al. 2009, Ding et al. 2002). The proposed
treatment will use gene therapy to target bone marrow stem cells, since all HIV target
cells (CD4+ Tells, macrophages, dendritic cells and even brain microglia) are derived
from bone marrow stem cells (Figure 3) (Bauer et al. 2000). If anti HIV genes are
inserted into bone marrow stem cells, all arising progeny will also carry these anti-HIV
genes. To accomplish this, bone marrow stem cells will be transduced with a lentiviral
vector that transfers a triple combination of anti-HIV genes. As learned from small
molecule anti-HIV drugs, for an effective defense against HIV, it is important to
simultaneously target multiple stages in the retroviral lifecycle because the relatively high
error rate of reverse transcription by the viral reverse transcriptase leads to frequent
mutations of the viral particles. The high rate of these mutations make it likely that the
virus will be able to escape a defense at only a single stage in its lifecycle, which is why
combinatory drug therapy has to be used. The same statement is true for gene therapy,
and therefore, three anti-HIV genes will be used. Each of the three anti-HIV genes will
interfere with HIV infection at a different stage of the HIV lifecycle: pre-entry, postentry/pre-integration and post-integration (Ding et al. 2002, Bauer et al. 2000, Mitsuyasu
et al. 2009).
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Figure 3. Bone Marrow Stem Cell Derived HIV Target Cells. Bone marrow stem cells
produce all blood cells, and at the same time maintain their stem cell pool by asymmetric
division. 1. After division, one cell of the pair remains a stem cell, and the other, after
undergoing multiple rounds of divisions produces blood cells, such as: 2. T cells, 3.
Macrophages, 4. Dendritic Cells, which are HIV target cells.
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Three anti-HIV genes have been engineered into one anti-HIV vector (Figure 4),
creating this triple combination anti-HIV lentiviral vector (Anderson et al. 2009,
Anderson et al. 2007). The first gene is an anti-CCR5 short-hairpin RNA (shRNA).
CCR5 is a chemokine receptor and is necessary for white blood cells to recognize
chemical gradients during the body’s immune response. It is also an essential co-receptor
for macrophage tropic HIV strains. Most strains of HIV are macrophage tropic. A natural
mutation occurring in approximately 1% of Caucasians, which causes the complete
deletion of the CCR5 chemokine receptor has no discernable phenotype due to receptor
redundancy, however, renders a person with a homozygous deletion resistant to HIV
infection. This phenomenon is the underlying reason for engineering an shRNA to knock
out the CCR5 receptor. The anti-CCR5 shRNA binds to the CCR5 mRNA and uses the
cell’s natural RNA interference mechanism to halt translation of the CCR5 receptor that
HIV needs to enter the host cell (Liu et al. 1996, Berger et al. 1999, Lee et al. 2002,
Martinez et al. 2002, Bai et al. 2000, Cordelier et al. 2003, An et al. 2007, Anderson et
al. 2007, Novina et al. 2002, Kumar et al. 2008, Huang et al. 1996, Naif et al. 2002,
Hutter et al. 2009, Fire et al. 1998, An et al. 2006, Castanotto et al. 2002, ter Brake et al.
2009).
The second gene, TRIM5α, is found in rhesus macaques and has been shown to
offer these old world monkeys natural immunity against HIV by inhibiting the uncoating
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Figure 4. Triple Combination Anti-HIV Vector. Vector diagram courtesy Dr. Joseph
Anderson, UC Davis. Into the pCCL lentiviral vector backbone, which contains the
reporter gene enhanced green fluorescent protein (EGFP) driven by the PGK promoter,
three anti HIV genes were cloned immediately after the RRE element: A Trim5alpha
protein driven by the MNDU3 promoter, a CCR5 shRNA driven by a U6 promoter, and a
TAR decoy, driven by another U6 promoter. The MNDU3 promoter is an excellent
promoter for protein expression, while the U6 promoters are designed to drive strong
RNA expression (shRNA and TAR decoy are both RNA based anti-HIV molecules).
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step of the viral capsid after entering the host cell. Humans also have a TRIM5α gene, but
HIV has evolved around it, therefore it cannot protect human cells from HIV infection.
The TRIM5α gene in thise triple combination vector is a re-engineered, chimeric version
of the rhesus macaque gene. The gene product is a humanized TRIM5α protein,
incorporating the 13 amino acid sequence providing HIV resistance from therhesus
macaque form into the human TRIM5α protein so the human body will not recognize this
protein as foreign and produce an immune reaction against it (Anderson et al. 2008,
Sremlau et al. 2008, Sawyer et al. 2005).
The third and final gene in the vector is a “transactivation response element”
(TAR) decoy. The HIV version of TAR binds to the viral protein, “transactivator of
transcription” (TAT), and in this complexed form leads to strong upregulation of HIV
transcription. The TAR decoy mimics the structure of the viral TAR and is able to bind
and occupy the viral TAT that is freely floating in the cell, so that TAT cannot reach
TAR, and therefore cannot aid in the initiation of efficient HIV transcription (Michienzi
et al. 2002, Kohn et al. 1999, Humeau et al. 2004, Bonyhadi et al. 1997).
The triple combination vector was shown to provide transduced Ghost-R5-X4-R3 cells,
which are sensitive indicator cells of HIV infection, with strong HIV resistance and
inhibition of viral replication. The triple combination anti-HIV vector has also been
tested on human hematopoietic stem cells engrafted into immunodeficient mice, where
they are expected to establish an HIV resistant, human immune system. The vector
worked as expected in the HIV mouse model (Anderson et al. 2009, Anderson et al.
2007).
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Figure 5. iPSC Based Stem Cell Gene Therapy for HIV. Figure courtesy Gerhard
Bauer, UC Davis. Induced pluripotent stem cells (iPSCs) from an HIV infected
donor are created, transduced with anti-HIV genes, selected and tested for safety,
differentiated into hematopoietic (= bone marrow) stem cells. These stem cells are
again highly tested for safety and efficacy, and if passed, are transplanted into the
autologous donor to enfgraft a new, completely HIV resistant immune system.
This new immune will then be able to control HIV as HIV will not find any new
target cells to grow in.
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The ultimate plan for patient treatment and an eventual functional cure for HIV
would be as follows (Figure 5):
1) Create induced pluripotent stem cells (iPSCs) from differentiated cells
from the patient to be treated.
2) Transduce the iPSCs with the triple combination vector.
3) Differentiate the iPSCs into hematopoietic stem cells.
4) Select for CD34+ and CD133+ cells and transplant into the patient.
5) The transplanted hematopoietic stem cells differentiate into functional
HIV resistant human blood cells in the patient.
This strategy will produce a virtually unlimited supply of HIV resistant,
hematopoietic stem cells. Although this treatment strategy is currently being developed, it
will not be available within the near future. However, an immediate clinical application
is now underway which will create anti-HIV gene transduced autologous bone marrow
(hematopoietic) stem cells from HIV infected individuals. Once the hematopoietic stem
cells are transduced and the vector is incorporated into the host cell genome, every cell
that arises from these stem cells will also have the vector and its anti-HIV genes in its
genome. Since all HIV targets cells arise from these hematopoietic stem cells, treated
patients will then have a very strong defense against HIV for the rest of their lives. These
transduced hematopoietic stem cells will then be given back to the patient in a bone
marrow transplantation setting to reconstitute the patient’s immune system with anti-HIV
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gene containing immune cells. It is anticipated that the anti-HIV gene containing immune
cells will have a selective survival advantage in the face of a viral load and provide the
patient with an increasing number of HIV resistant immune cells. Over time, this could
lead to a fully HIV resistant immune system that might be able to control HIV infection
without the need of HAART.
My role within the broader project is to produce the clinical grade lentiviral vector
on a large scale in the Good Manufacturing Practice facility. Briefly, to produce a safe,
replication incompetent HIV based gene therapy vector (Figure 6), the inherent capability
of HIV, which is a lentivirus is being used to transfer a gene of interest, in our case, a
triple combination of anti-HIV genes into a target cell, the bone marrow stem cell and
integrate it there permanently. The lentiviral particle is a re-engineered H IV particle. It
follows the same life cycle of HIV, it attaches to the target cell, reverse transcribes its
RNA into DNA, transports it into the nucleus and permanently integrates it into the DNA
of the target cell. The genetic information how to produce the HIV structural proteins and
the viral enzymes is still available, however, the information how to package the HIV
RNA into the assembled viral particle has been completely removed. This information is
encoded in a region of the viral genetic information called the “psi” region. Additionally,
the information how to produce endogenous HIV envelope is deleted and replaced with
the genetic information for the Vesicular Stomatitis Virus-G (VSV-G) envelope protein
(Varmus et al. 1997, Naldini et al. 1996, Anderson et al. 2009). This is done to make the
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Figure 6. Generation of a Lentiviral Vector. A psi-region deleted packaging plasmid only
carrying the absolute minimum of endogenous HIV genes necessary to produce a viral particle
and the viral enzymes called “packaging plasmid” (1), an envelope plasmid coding for the
Vesicular Stomatitis Virus G protein (2) and a gene of interest carrying the psi-region in order
to be packaged into the viral particle are transfected into the producer cell, a 293T cell (4).
Together, these plasmids code for a viral particle that does not contain any HIV RNA, but only
RNA of the gene of interest.
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virus particle “pan tropic”, which means it can infect all mammalian cells, facilitating
better gene transfer into target cells. Finally, the therapeutic gene of interest, which needs
to be transferred into the target cell is engineered. This gene mimics the HIV RNA, but
only in the terminal regions of the RNA, which are called “Long Terminal Repeats”
(LTRs). In between the LTRs, a maximum of 10-15 kilobases of genetic information can
be cloned into. No other essential information how to make HIV is present. The most
important addition, however, is the insertion of the psi region into the therapeutic gene of
interest, which now allows the therapeutic gene to be packaged into the viral particle
during the particle assembly process (Figure 6). Theoretically, all necessary genes to
make a gene therapy vector could be cloned into one plasmid, but to prevent any
accidental gene recombination in the producer cells leading to replication competent
particles, the lentiviral genetic components making up the final vector are separated on
three plasmids and are transiently transfected into the producer cell separately (Figure 6).
The producer cell originates from a human embryonic kidney (HEK) cell line called
293T, which has been shown to be highly transfectable with vector DNA, yielding
excellent titer. The final viral particle derived from this producer cell is called the “gene
therapy vector”. The producer cell will produce vector particles transiently, since the
vector plasmids will be lost over the long run, and additionally, the VSV-G envelope is
lytic and therefore destroys the producer cells after several days. A new vector
preparation therefore always requires a new transient transfection setup (Sheridan 2011,
Varmus et al. 1997, Naldini et al. 1996, Anderson et al. 2009, Ding et al. 2002, Anderson
et al. 2007, Humeau et al. 2004).
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The initial vector has already been tested on human hematopoietic stem cells
engrafted into immunodeficient mice to make sure it is safe and inhibits HIV infection.
Once the safety and efficacy of the vector had been confirmed, it was determined that it
had to be produced in large amounts for possible clinical applications. In addition, the
clinical grade vector will also need to be evaluated for sterility, absence of endotoxin and
absence of any replication competent virus (Anderson et al. 2009, Anderson et al. 2007).
This project requires me to first grow the producer cell line, the HEK 293T cell
line in large quantities, transfect the expanded cultures with 3 plasmids encoding the
vector gag and pol sequences (the structural proteins and the polymerases), the vector
envelope, which is the VSV-G protein, and the triple combination anti–HIV gene. After
this transfection step, vector supernatant is collected and then tested for sterility,
endotoxin and absence of replication competent lentivirus (RCL), all while complying
with Good Manufacturing Practice standards which can be found in: 21 Code of Federal
Regulations parts 210 and 211 (21 CFR 210 and 211). Good Manufacturing Practice is
enforced by the US Food and Drug Administration (FDA) and is intended to ensure the
quality and safety of pharmaceutical products for human administration. Important
aspects of Good Manufacturing Practice manufacturing of this specific product are
further discussed in the discussion section.
In addition to providing possible treatments for infectious diseases, such as HIV,
gene therapy may present the medical field with a permanent cure for genetic diseases,
such as epidermolysis bullosa (EB), by way of induced pluripotent stem cell (iPSC)
manipulation. The treatment of EB through the Good Manufacturing Practice
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manufacturing of iPSCs from EB patient fibroblasts, their gene correction through
homologous recombination, the differentiation of gene corrected iPSCs into keratinocytes
and the manufacturing of gene corrected, patient specific dermal grafts for transplantation
onto open wounds is another project being actively pursued in the UC Davis Good
Manufacturing Practice facility.
(iPSCs) are pluripotent cells created from any type of cell by reverting them back to
an embryonic stem cell-like state. This pluripotency is created by integrating four genes
into the cell’s genome known to revert cells back to a pluripotent state. The four genes
are: Oct4, Sox2, c-Myc and Klf4, combined into one DNA vector which is then packaged
into a replication incompetent lentiviral vector capable of delivering the DNA to cells.
Human skin fibroblasts can then be transduced with this lentiviral vector which inserts
the early acting factors into the genome of the fibroblasts. Expression of these early
acting factors then revert differentiated fibroblasts into pluripotent cells. The pluripotent
characteristics of iPSCs allow them to be differentiated into any almost cell type. This
quality, combined with the ability to manipulate these cells’ genomes using gene therapy,
provides researchers with an immensely powerful new tool to aid in research (Sheridan
2011, Tolar et al. 2010).
In order for these lentiviral vectors to be used in clinical research, they must be
produced at a titer of at least 107 particles/mL. During my internship at the UC Davis
Institute for Regenerative Cures, I was successful in producing both the anti-HIV
lentiviral vector and the iPSC producing lentiviral vector at a titer in the 109 particles/mL
range using a lipofection transfection procedure followed by spin filtration to concentrate
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the viral particles. I also produced a third experimental lentiviral vector containing antisickle genes for use in a treatment for sickle-cell anemia. This vector, however, yielded
approximately 107 particles/mL only, using the same methods as the other two vectors. It
was confirmed with the vector developer that this was the expected titer due to the size of
the anti-sickling gene (Anderson et al. 2009). A fourth experimental vector, another antiHIV gene vector produced for a private company only yielded approximately 106
particles/mL. This outcome was unexpected, and will be discussed in more detail in the
discussion section.
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MATERIALS AND METHODS
In order to create a stock of Good Manufacturing Practice quality 293T human
embryonic kidney (HEK) cells for future use in various procedures, a 200 vial master cell
bank of these 293T cells was created. A 1mL vial of 293T cells was ordered from ATCC.
The cells were split into two 225cm3 mL flasks and were grown in D10 media (high
glucose Dulbecco’s Modified Eagle Medium (DMEM), 10% defined Fetal Bovine serum
(FBS) and 1% Glutamax L-glutamine). When the cells had reached 70-80% confluency,
they were harvested using trypzean. Cells should not be allowed to become too confluent
because they might become senescent. Trypzean is the plant version of the animal
protease trypsin and was used in this procedure to keep the cells in a xeno-free
environment. Once the cells were lifted from the flasks, they were suspended in freezing
media (90% defined FBS and 10% dimethyl sulfoxide (DMSO)) and aliquoted in 1mL
portions into ten cryovials at a concentration of approximately 5x106 cells/mL. The cells
were then frozen down to -90°C using a controlled rate freezer and then stored in the
vapor phase of a liquid nitrogen freezer at -120°C or below. Once the cells are suspended
in the freezing media, they should begin the freezing process as quickly as possible. The
DMSO allows the cells to use a conserved sulfur-based metabolic pathway so the cells
can still metabolize when oxygen is not available during the freezing process, but the
DMSO can become toxic to the cells if they are suspended in it too long without being
frozen down. This 10 vial cell bank was created to provide a stock of passage 1 293T
cells for future use. Following the creation of this 10 vial cell bank, one vial was thawed
into two 225cm3 flasks and was grown out into sixteen 225cm3 flasks using the same
26
method as before until those sixteen flasks were 70-80% confluent. The cells were then
collected and frozen down in 1mL aliquots into 200 vials at a concentration of
approximately 4.5x106 cells/mL using the same methods as described above. This
provided us with a 220 vial master cell bank of Good Manufacturing Practice quality,
passage 5, 293T cells. All procedures described were performed in the Good
Manufacturing Practice facility under Good Manufacturing Practice conditions.
To produce concentrated stocks of our anti-HIV vector, anti-sickling vector and
our IPSC induction vector, one vial of 293T cells from the passage 5 master cell bank
was thawed and grown until four 225cm3 flasks were about 70-80% confluent. Once a
confluency of 70-80% was reached, the cells were lifted using trypzean, counted and
25x106 cells were plated back into each of the four 225cm3flasks. 22mL of D10 media
was used in each of the flasks. Approximately 24 hours after plating the 25x106 cells, in
each of four 15mL conical tubes (one per 225cm3 flask of cells), 150μL of Trans-IT
lipofection reagent was added to 3mL of serum free media (incomplete DMEM). The
mixture was mixed by pipetting up and down once and then incubated at room
temperature for 20 minutes. While the lipofection mix was incubating, in each of four
1.5mL microcentrifuge tubes, 25μg of pCMVdR8.9 packaging plasmid, 5μg of VSVG
envelope plasmid and 25μg of vector DNA of interest were mixed together; this is the
DNA mix. After the 20 minute incubation of the lipofection mix, each 1.5mL
microcentrifuge tube of DNA mix was added to a different 15mL conical tube of
lipofection mix. The mixture was pipette up and down once then incubated at room
temperature for 30 minutes. After the 30 minute incubation, the contents of each 15mL
27
conical tube was transferred, dropwise, into a different 225cm3 flask of 293T cells.
Approximately 24 hours later, the media in each 225cm3 flask was replaced with 30mL
of UltraCulture serum free media. 48 hours after the media change the viral supernatant
was removed from each flask and transferred to a 50mL conical tube where it was
centrifuged at 3,800 rpm for 5 minutes. The supernatant was then transferred to Centricon
plus-70 filtration units and centrifuged at 3,800 rpm for 35 minutes. The retentate was
collected and centrifuged at 1,100 rpm for 5 minutes and the resulting viral supernatant
was filtered through 0.45mm Spin-X tubes. The viral supernatant was then aliquoted in
30μL portions into cryovials and stored at -80°C.
To determine the viral load in the concentrated, frozen aliquots, 5x105 293T cells
were plated in 2mL of D10 media in each well of a 6-well plate. 24 hours later, the media
was removed from each well and replaced with 1mL of D10 media + 1μL of protamine
sulfate per well. A 100x vector dilution was made by adding 10μL of thawed vector into
990μL of media. Then a 10,000x dilution was made by adding 10μL of the 100x vector
dilution into 990μL of media. Further dilutions were made by adding different amounts
of these dilutions to the media on the cells. A 1x, 10x, 100x, 1,000x, and 10,000x dilution
were created as well as a well with no vector for the negative control. The cells were
incubated at 37°C and transduced overnight. 24 hours later, the media in each well was
replaced with 2mL of D10 media. About 48 hours after the media change, the cells in
each well were lifted using trypsin and either analyzed directly by flow cytometry or
saved for qPCR.
28
Cells that were transduced using a vector containing GFP were analyzed by flow
cytometry. After the cells were lifted with trypsin, they were transferred to a
corresponding FACs plastic tube and analyzed by the flow cytometer for the presence of
GFP. Vectors analyzed by flow cytometry were the anti-HIV triple combination vector.
Cells that were transduced using a vector that did not contain GFP were analyzed
for viral integration by qPCR. After the cells were lifted with trypsin, the cells were
transferred to corresponding 1.5mL microcentrifuge tubes and DNA extraction was
performed using the Promega DNA extraction kit #A1620. DNA from each dilution was
diluted to 66ng/μL. 5μL of each DNA dilution were added to 7μL of the reaction master
mix for a total reaction volume of 12μL in each well of a 96 well PCR plate. The master
mix consisted of a 2:1 primer to probe mix added to a volume of TaqMan PCR master
mix that was ten times the amount of the primer volume plus enough water to bring the
total reaction volume to 12μL in each well. The primers were specific to the WPRE
sequence: Forward: 5’-CCG TTG TCA GGC AAC GTG-3’. Reverse: 5’-AGC TGA
CAG GTG GTG GCA AT-3’ and the probe used was FAM/TAMRA; sequence: 5’FAM-TGC TGA AAC ATT CAC CTT CCA TGC AGA-TAMRA-3’. Both the primers
and probe were ordered from IDT Technologies. The master mix used was the TaqMan
Universal PCR Master Mix, No AmpErase UNG from Applied Biosystems. Vectors
analyzed by qPCR were the anti-sickle vector and the IPSC induction vector. The
plasmid used for the standard curve was the pCCL-CMNDU3-eGFP-WPRE plasmid
from which dilutions of 102-106 were made. All reactions including the standard curve
were run in duplicate. PCR conditions were one incubation at 50°C for 2 minutes then an
29
incubation at 95°C for 10 minutes. This was followed by 40 cycles of incubation at 95°C
for 15 seconds then at 60°C for 1 minute.
30
RESULTS
New 293T cells from the American Type Culture Collection (ATCC) were
expanded into sixteen 225cm2 flasks. The expansion required several splits of the original
cell seed stock, which was received frozen. An initial, low passage, small stock cell bank
was established; this also helped to adapt the new 293T cells to our culture conditions.
After culture adaptation, 10 vials with low passage 293T cells was frozen using the
controlled freezer, and the remainder of the cultured cells was adapted to large scale
expansion. One 225cm2 flask was used initially, from which an expansion into 16 225cm2
flasks was attempted. Successful expansion with multiple splits into 4, 8 and 16 flasks
could be performed. The expansion process took approximately 14 days. Visual
observation was performed daily, and cell confluency state below 80% was maintained at
all times. After trypsinization, washing and pooling of all harvested cells, a final cell
number of more than 900 million cells total was achieved; 16 flasks proved to be enough
to provide us with the needed 200 vial master cell bank of 293T producer cells for use in
vector production. Each individual cryovial received approximately 4.5x106 million cells,
and all vials were filled using aseptic technique. The cells were frozen to -90°C using a
controlled rate freezer with a temperature drop rate of 1deg Celsius per minute and then
stored at or below -120°C in the vapor phase of liquid nitrogen. To test the quality of the
master cell bank, a sample of 1% (2 vials) of the master cell bank randomly drawn from
the stored vials was evaluated for viability, sterility and endotoxin. The cell viability
count upon thawing of each vial was greater than 98% as measured by trypan blue
exclusion. A validated USP 14 day sterility assay and a validated endotoxin assay were
31
performed on the thawed samples from the master cell bank. These specific assays are
required in the context of Good Manufacturing Practice for the establishment of a master
cell bank for clinical grade gene therapy vector manufacturing. The validated assays were
performed in the quality control testing lab of the Good Manufacturing Practice facility.
The tested vials were confirmed to be sterile and free from endotoxin.
The new master cell bank was then used for lentiviral vector manufacturing.
Although my project only involved the manufacturing of gene therapy vector for HIV
gene therapy and induction of pluripotent stem cells, I also manufactured test batches of
two other lentiviral vectors. The additional vectors were another anti-HIV gene vector
from a company called Calimmune, and an anti-sickling vector for gene therapy of sickle
cell anemia, commissioned by UCLA. Good Manufacturing Practice vector
manufacturing poses a challenge, since maximum titer at a large scale is required to yield
reasonable transduction efficiencies in the target cells. Not all therapeutic vectors easily
yield high titer. Titer is dependent on many parameters, but first and foremost on good
construction of the vector plasmid which determines the strength of the packaging signal
and efficient packaging.
All lentiviral vectors were manufactured in identical fashion. An initial vector
manufacturing run was used as an engineering run, after which the method of vector
manufacturing could be standardized and written up as a Standard Operating Procedure.
Production of both the anti-HIV vector and the iPSC-induction vector yielded 25 vials
containing 25μL of concentrated supernatant of each vector. The vials are stored at -80
32
deg Celsius. Production of the additional lentiviral vectors for both Calimmune and
UCLA also yielded 25 vials of concentrated vector.
To determine the titer of the lentiviral vectors, two methods of titration were used:
The anti-HIV vector contained GFP so its titer could be determined by flow cytometry
analysis while the iPSC-induction vector was tittered using quantitative Polymerase
Chain Reachtion (qPCR). Briefly, for the flow cytometry analysis of the anti-HIV
vectors, 293 cells were transduced with vector dilutions, the number of GFP expressing
cells were enumerated, and the titer was calculated according to the vector dilution and
numbers of GFP positive cells. For qPCR a similar method was applied, however, the
GFP readout was replaced with readout for the signal of the WPRE element in the vector.
GPF titers deliver a robust result with less than 5% deviation in vector titer. qPCR titer
results are less robust and produce a larger error.
Vector Titer Results (all readouts in transducing units per mL):
GFP readout:
Anti HIV Triple-combo vector, manufactured 7/17/10: 1x108
Anti HIV Triple-combo vector, manufactured 8/14/10: 5x109
Anti HIV Triple-combo vector, manufactured 11/30/10: 3x109
qPCR readout:
Stanford iPSC induction vector, 1/14/11: 1x105 – 7x109
33
Other project vectors (qPCR readout):
CalImmune vector, manufactured 10/22/10: 3x105 – 2x106
CalImmune vector, manufactured 11/30/10: 5x105 – 4x106
UCLA vector, manufactured 10/22/10: 7x106 – 4x108
UCLA vector, manufactured 1/14/11: 1x106 – 9x108
All titrations were performed in duplicates.
Figure 7 shows a graphic comparison of the vector titers. It is clear to see that the
Calimmune vector yielded, in a repeatable fashion, the lowest titer of all vectors
manufactured. The triple combination anti-HIV vector repeatedly resulted in the best
titer, closely followed by the Stanford vector. The large error bar pointing towards a
lower titer in the Stanford vector could not be confirmed to be accurate in subsequent
transduction experiments; therefore the higher titer indicated by the bar is assumed to be
correct. The UCLA vector resulted in an intermediate titer. An additional optimization
run for the UCLA vector has been performed using multiple timed supernatant
collections. Titer results of these collections are not yet available.
The Stanford vector was only produced once, another production run is planned.
The triple combination anti-HIV vector was used by the HIV group at the UC Davis Stem
Cell program to transduce hematopoietic stem cells, and was also used to transduce
induced pluripotent stem cells. Both hematopoietic stem cells and pluripotent stem cells
were then differentiated into mature blood cells, which could be demonstrated to be
34
resistant to HIV infection. Figure 8 shows an induced pluripotent stem cell colony that
was transduced with the anti-HIV vector.
The excellent titer of the iPSC induction vector led to several experiments within
the Good Manufacturing Practice facility to manufacture Good Manufacturing Practice
grade induced pluripotent stem cells from skin fibroblasts. Briefly, the vector was diluted
to a multiplicity of infecton of 10, 20, 30 and 40. (Multiplicity of infection means the
number of transducing vector particles per cell.) Skin fibroblasts that had been grown
under Good Manufacturing Practice conditions were transduced overnight with the iPSC
induction vector, next day they were plated onto irradiate human foreskin fibroblast
feeder cells. After about 8-10 days of growth, suitable iPSC colonies were mechanically
dissected and transferred onto new irradiated human feeders. Established colonies were
then enumerated and photographed. Figure 9, as an example, shows two iPSC colonies
derived using the Stanford iPSC induction vector.
Vector Titer (particles/ml)
35
1.00E+10
1.00E+09
1.00E+08
1.00E+07
1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
Replicate 1
Replicate 2
Replicate 3
Figure 7. Comparison of Lentiviral Vector Titers. Y axis: Titer in transducing units per mL.
X axis: Lentiviral vector preparations. Calimmune, UCLA and Stanford vectors were titred
using qPCR readout, the Triple Combination anti-HIV vector was tittered using GPF flow
cytometry readout. There are no error bars in the GFP readout as the readout error is less
than 5%.
36
Figure 8. Transduced iPSC Colony Using the Anti-HIV Gene Vector. a. Fluorescent image
(GFP). b. Phase contrast image. c. Overlaid fluorescent and phase contrast image. All cells
within the colony are transduced and express GFP. Photos courtesy Amal Kambal, UC Davis.
37
Figure 9. Good Manufacturing Practice Grade iPSC Colonies Using the Stanford iPSC
Induction Vector. a. 8 day, post-dissection iPSC colony at the edge of the culture well, b. 8 day
post dissection iPSC colony in the center of the culture well. Photos courtesy Gerhard Bauer,
UC Davis.
38
DISCUSSION
My work in the UC Davis Good Manufacturing practice facility allowed me to
learn and apply the principles of Good Manufacturing Practice in a stem cell research
setting. Good Manufacturing Practice is a national standard established to produce safe
and efficacious pharmaceuticals. This standard has recently also been applied to cellular
therapeutics, and all associated materials needed to produce such cellular therapeutics, in
our case, lentiviral vector used to transduce stem cells. In order to use a stem cell product
clinically, it must therefore be assured that the product has been produced in a Good
Manufacturing Practice compliant manner, and is safe and efficacious. Such a product
has to be manufactured (which is the term the FDA uses for any clinical grade production
of therapeutics) in aclean-room environment, a Good Manufacturing Practice laboratory.
This laboratory features a controlled environment, that means that the air in the
manufacturing rooms for the cellular therapy product is free of particles (fungus, spores,
etc.) which could possibly contaminate the product, and that temperature and humidity
are kept constant. Personnel working in such a facility have to gown up, since they also
should not introduce a contamination risk to the product. Additionally, if a gene therapy
vector such as a lentiviral vector should be manufactured under Good Manufacturing
Practice conditions, the clean room needs to be under negative pressure to contain a
possible vector aerosol that might be introduced during the spinning and concentration
process. Full gowning of personnel, which includes face masks also protects personnel
from possible aerosols. It therefore becomes clear that a Good Manufacturing Practice
facility is a highly sophisticated, technically complicated laboratory that needs to follow
39
strict building and operational standards. The UC Davis Good Manufacturing Practice
facility has been designed to meet and exceed the federal standards, and has some unique
features, such as switchable room pressurization to produce negative pressure
manufacturing rooms for the manufacturing of gene therapy vectors.
Before I could even start my project, I therefore had to receive training in how to
work in such an environment, and how to quality control this environment and the
manufactured product. The rules of Good Manufacturing Practice manufacturing are: All
manufacturing steps and the facility itself have to be in a state of control, all processes are
written down in Standard Operating Procedures (SOPs), the final product undergoes
Quality Control (QC) testing and a final review by the Quality Assurance (QA) unit.
My project started with the development of a Good Manufacturing Practice grade,
triple combination anti-HIV gene lentiviral vector. First, engineering runs needed to be
performed, as the Good Manufacturing Practice facility had just opened, and this
lentiviral vector manufacturing project was one of the first projects to be undertaken in
this new facility. During the engineering runs, the methods had to be refined, and the
SOPs needed to be developed. In Good Manufacturing Practice manufacturing everything
revolves around SOPs. The technician needs to adhere to these SOPs strictly to always
manufacture a reproducible product, and the QC unit supervises this step.
An important obstacle that needed to be overcome in the beginning was the
generation of the HEK 293 T master cell bank. For the engineering runs, 293 T cells from
the adjacent translational laboratory were used. For clinical purposes, however,
laboratory grade cells cannot be used. Therefore, new cells had to be ordered from the
40
American Type Culture Collection (ATCC) with a certificate of analysis. Then I needed
to expand these cells greatly to produce a 200 vial master cell bank. It took several weeks
to just expand these cells in large tissue culture flasks to arrive at a quantity sufficient for
the 200 vial master cell bank. Additionally, no contamination was allowed in this
expansion process. After establishing this master cell bank, a working cell bank was
generated from the master cell bank, which was then tested for sterility and the absence
of endotoxin. Only upon receiving the test result clearing the working cell bank it was
safe to manufacture clinical grade lentiviral triple combination anti-HIV gene vector.
This clinical grade vector from the new master cell bank compared extremely well in titer
(109/mL) with vector from the engineering run and now allows the HIV team to move
forward, with confidence, initiating clinical trials for stem cell gene therapy for HIV. An
application to the NIH Recombinant DNA Advisory Committee (RAC) is currently being
drafted as the first step for approval of this clinical trial, with the final Investigational
New Drug Application (IND) being submitted to the FDA.
As the Good Manufacturing Practice manufacturing of a high titer, triple
combination anti-HIV gene vector was successful, other vector manufacturing projects
were moved forward. The vector for the iPSC project had been obtained from
collaborators at Stanford, who had not manufactured clinical grade vector before. Often,
clinical grade vector is associated with lower titer, since Good Manufacturing Practice
vector production has to follow stringent procedures that emphasize safety (freedom from
contamination), but not maximum titer. In order to obtain successful iPSC induction,
however, a high transducing titer is essential. Using the optimized vector manufacturing
41
protocols I developed, I was able to also achieve the needed high titer in this vector
manufacturing run. Good Manufacturing Practice grade iPSCs have now been produced
twice using this Good Manufacturing Practice grade lentiviral vector preparation.
However, problems that arose with the additional vectors I manufactured should
also be mentioned. The anti-HIV gene vector commissioned by a private company
yielded disappointing titer upon the first try; therefore, I manufactured a subsequent lot.
To make sure that the manufacturing process worked well, I manufactured our own
combination anti-HIV vector in parallel. Our own vector yielded a titer in the 109 range,
while the company vector again only yielded a titer in the 106 range. As the appropriate
control was performed in parallel and yielded excellent titer, an error in the vector
manufacturing process could be ruled out. We therefore must assume that the company
anti-HIV gene transfer vector itself was at fault. It has been known for a while that an
anti-HIV vector may inhibit its own packaging due to the inherent anti-HIV effect, which
will also be exerted upon the packaging cell, as the genetic information of HIV is used to
produce the vector particle. Our own anti-HIV vector has been optimized to overcome
this problem. We do not know, however, if the company vector has undergone the same
optimization. Additionally, the way the therapeutic vector was cloned by the company is
also not known; often, optimization steps need to be performed to make packaging more
efficient, as the packaging signal can be dependent on its precise location within the
vector. We also do not know if such optimization steps were undertaken by the company.
Since we were not paid to perform any further optimization on this vector, the project had
to be put aside.
42
The other lentiviral vector I manufactured for UCLA was an anti-sickling gene
vector intended for a stem cell gene therapy approach to treat sickle cell anemia. As with
the previous vector, I performed an initial vector manufacturing run which yielded a
vector titer in the 10e7 range. As we are used to higher vector titer, which we routinely
achieve with our combination anti-HIV gene vector, we decided that another vector lot
should be manufactured. Again, to rule out a problem with vector manufacturing, our
own anti-HIV gene combination vector was manufactured in parallel, yielding titer
results in the high 109 range, while the anti-sickling vector again yielded a titer in the 107
range. We reported these results to UCLA, and also sent aliquots of the vector to UCLA
for their own tittering, again confirming our results. We then were informed by the
UCLA investigator that the titer result we had achieved was the usual titer results
obtained from laboratory grade anti-sickling vector preparations. It is thought that the
large therapeutic gene present in the vector, which almost reaches the carrying capacity
of the lentiviral vector, may be responsible for the lower titer. To improve vector yield, I
therefore manufactured another vector lot, and took several vector collections at different
time intervals to find out if we can improve titer and also the number of useful vector
collections by careful collection timing.
In order to reliably produce lentiviral vector in a Good Manufacturing Practice
fashion, high precision technical work with complete adherence to SOPs is required.
Over the last year, I have been able to master the skills to successfully work in a
sophisticated Good Manufacturing Practice environment. My ability to reliably produce
high titer vector that is safe and efficacious could be demonstrated in the safety and
43
translational studies performed with our combination anti-HIV vector and the iPSC
induction vector. Additionally, it could be demonstrated that lower titer results obtained
with different vectors commissioned for Good Manufacturing Practice production by
outside companies were due to inherent vector problems, and not the manufacturing
process and techniques I developed.
During this last year, I have also made a valuable contribution to translational
medicine. The combination anti-HIV lentiviral vector I manufactured will be used in a
clinical trial for the transfer of anti-HIV genes into patient hematopoietic stem cells to
achieve an HIV resistant immune system; the iPSC induction vector will be used to
generate iPSCs from patients with Epidermolysis Bullosa, eventually resulting in gene
corrected dermal sheets for grafting onto these patients. Hopefully soon my work will be
providing patients with the much needed clinical benefit this research has been designed
to achieve.
44
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