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Molecular therapeutics inhematology: gene therapy 333
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likely more immune- privileged site (the bladder) allows
repeat administration of the treatment.
At this time, the most widely employed adenoviralbased therapies are COVID- 19 vaccines. Five different
adenoviral- based SARS- Cov- 2 vaccines have emerged;
CanSino’s Ad5- nCoV (human Ad5), Janssen- Johnson &
Johnson’s Jcovden (human Ad26), Oxford- AstraZeneca’s
Covishield and Vaxzevria (chimpanzee AdY25), and
Gamaleya Institute’s Sputnik- V (human Ad26 for the first
dose and human Ad5 for the second dose). Each employ
slightly differing codon- optimized cDNA sequences and
their own take on the signal sequence used to generate the
best SARS- Cov- 2 spike expression and protein conformation. While not integrating into the patients’ DNA, these
adenoviral- delivered cDNA exist episomally in the vaccine, and produce SARS- Cov- 2 spike mRNA and protein
to elicit an immune response. It is believed that the original dose likely leads to a longer- term initial vaccination,
but for the reasons mentioned above re- dosing and booster
applications may have increased side effects and are
unlikely to be as effective as with the other non- adenoviral
based COVID- 19 vaccines.
Adeno- associated virus gene transfer
Versions of the human parvovirus, AAV, are also being used
more frequently as vehicles for therapeutic gene transfer.
AAVs are thought to be non- pathogenic in humans and
establish a latent infection in the absence of adenovirus itself
or helper functions provided by other viruses. Wild- type
AAV integrates at some frequency into a specific site on
human chromosome 19q13.3–qter, which appears to be a
relatively benign location. Only the AAV inverted terminal
repeats are required as transcriptional units for recombinant
AAV vectors, which allows about 4 kb for foreign inserts,
given the size of the parental viral genome. However, both
the efficiency and specificity of integration are lost without
the wildfrequency. Due to the risk of insertional mutagenesis posed
by integrating vectors, this, along with low immunogenicity,
has become a major selling point for AAV gene therapy.
With that said, there have been reports of AAV delivery
resulting in hepatocellular carcinoma formation in mice,
and wild- type AAV2 has been found to integrate near
andpotentially dis- regulate oncogenes in human hepatocellular carcinomas. Thus, further study on recombinant AAV
gene therapy vector integration is warranted. AAV vectors
also carry a small genetic payload, leaving them clinically
useful only for the delivery of relatively small transgenes.
As usually produced, AAVs have titers of approximately
106 particles/mL and can be concentrated to greater than
109 particles/mL, although in each preparation many
type genes; recombinant AAV integrates at a low
non- infectious particles are generated. Human CD34+
hematopoietic stem/progenitor cells have been transduced
with recombinant AAV vectors, with up to 80% of colonyforming units (CFU) carrying the transgene. Interestingly,
the optimal use of recombinant AAV- based gene transfer
vectors to impact the hematopoietic system may be in
secondary manifestations (as follows).
In a landmark study, Kay and colleagues injected recombinant AAV vectors that engineered expression of factor IX
(F.IX) into skeletal muscles of severely afflicted hemophilia B
patients. Long- term vector persistence was observed in that
study as were slight increases in the circulating levels of the
corrective proenzyme. Subsequent studies aimed to improve
on the limited F.IX expression seen in this early AAV work.
Using a new AAV serotype (AAV8) and an improved
transgene design, a dose- escalation study infusing F.IX
AAV8into peripheral veins was conducted. F.IX expression
was stable3 years after infusion in most patients and averaged 5.1% of normal in the high- dose group. The treatment
was well- tolerated. However, it should be noted that some
ofthese patients were given an immunosuppressive corticosteroid 4–8 weeks post- treatment. Importantly, most of
the high- dose patients were able to reduce their exogenous
F.IX treatment post- gene therapy. This pioneering work led
to Hemgenix’s approval by the FDA in November 2022,
and the EMA in February 2023. Hemgenix is a single IV
infusion of an AAV5 engineering F.IX, which leads to liver
transduction and increased circulating F.IX. Hemgenix’s pivotal study enrolled 54men with hemophilia B and reported
increased F.IX levels, a decreased need for routine F.IX
replacement prophylaxis, and a 54% reduction in annualized
bleeding rate.
Considerable work has also gone into investigating the
use of AAV to treat type 2 Leber congenital amaurosis
(LCA). LCA is an inherited retinal dystrophy that causes
loss of vision at an early age. In three independent stage I/II
trials using Luxturna (voretigene neparvovec, hRPE65), an
AAV2 construct, a total of 20 patients were treated by subretinal vector administration. In all three trials, all patients
experienced improvements in visual sensitivity peaking
roughly a year post-
treatment. In two of the three trials, the
patients began to lose the AAV- confirmed benefit after
2–3years. However, sustained improvement in their condition was noted in the third trial. A following phase III study
reported that 65% of participants displayed maximal
improvement in the trial’s primary endpoint: bilateral multiluminance mobility testing, 1 year after treatment. On the
back of this study, in 2017, Luxterna became the first AAV
gene therapy approved by the FDA, with EMA approval
following in 2018.
In α1- antitrypsin (AAT) deficiency, mutations in the
SERPINA1 gene lead to reduced liver secretion of AAT and
impaired anti- protease activity in the lung leading to
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334 Molecular Hematology
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early- onset pulmonary emphysema. In an early trial, AAV2
encoding the AAT cDNA sequence was injected intramuscularly into the upper arm of 12 patients at various doses, all of
which were below comparable levels used in rodent studies.
Serum AAT levels were found to be above background in
only 1 of the 12 subjects dosed, which was rather unsurprising given the relatively low dosage used. A follow- up trial
made use of an AAV1 vector as it displayed 100- fold higher
hAAT serum levels than that seen with AAV2 constructs in
murine muscle transduction models. This follow- up AAV1
study resulted in detectable AAT in the serum of almost all
patients, and they maintained subtherapeutic levels of this
factor for at least 90 days and up to 1year post- treatment. A
phase 2 trial followed aiming to employ a dose escalation to
achieve therapeutic transgene levels. To reach the maximum
dose, AAV was delivered in 100 separate intramuscular
injections. A linear relationship between dose and serum
AAT levels was noted; however, even at the maximum dosage serum AAT levels were still below a therapeutically relevant level.
Zolgensma (AVXS- 101) was approved in the United
States (2019) and Europe (2020) as a treatment for children
with spinal muscular atrophy type 1 (SMA1) with an SMN1
gene disruption. SMA1 is caused by low levels of the survival motor neuron protein and affects all muscles in the
body. Zolgensma is a single intravenous peripheral limb
vein infusion of an AAV9 vector encoding SMN1. A Phase
1 trial evaluated Zolgensma in 15infants; finding improved
survival as well as improved motor skills especially in the
higher dose cohorts. A follow- up Phase 3 trial evaluated
22infants. Zolgensma infusion increased survival, reduced
the need for a permanent ventilator to breathe, and
improved motor skills including ability to sit without support and stand and walk without assistance. Forty- one percent of the patients metall three criteria for ability to thrive
at 18 months of age,
Attempts at using DNA- editing (above) have begun in
the clinic, with one study using AAV to deliver an antiCCR5 ZFN into 12 HIV patients. Years following treatment, the gene- modified T cells could be detected in
circulation, but only at low levels. TALENs have been used
clinically to disrupt T- cell receptor alpha (TRAC), glycoprotein CS1, and CD52 as well as several cancer- specific
antigens. CRISPR- Cas has been used in a growing number
of trials to impart immunomodulating activity, such as by
deleting TRAC, T- cell receptor beta (TRBC), and programmed cell death protein 1 (PD- 1) in engineered autologous T lymphocytes.
Many other AAV vectors are currently being evaluated in
both preclinical and clinical proof- of- concept studies.
Importantly, many emerging gene editing trials including
CRISPR- based studies, use AAV delivery or a combination
of AAV and non- viral delivery methods.
Herpes simplex virus gene transfer
To date, there has been one approved modified herpes simplex virus used as a gene transfer vector. Imlygic is an oncolytic herpes simplex virus 1 used to treat melanoma patients
for whom surgical intervention is not appropriate and that
have tumors that can be directly injected. It was one of the
earliest approved oncolytic immunotherapies gaining
approval in both the United States and EU in 2015. Imlygic
has been genetically engineered to express GM- CSF, but to
lack infected cell protein 34.5 and 47 (ICP- 34.5 and ICP- 47).
Imlygic targets both cancerous and healthy cells, but without
ICP- 34.5 it cannot productively replicate in healthy cells.
However, a melanoma cell’s stress response is commonly disrupted, often allowing Imlygic to still replicate, until the
melanoma cell swells. Following cell death, the replicated
Imlygic can infect nearby cells to repeat the process. The
encoded GM- CSF is released when cancer cells burst, attracting immune cells capable of presenting melanoma antigens
to cytotoxic T cells, hopefully creating a cascading antimelanoma immune response. ICP- 47 normally suppresses
the immune response to the virus. ICP- 47 is removed– as
part of Imlygic’s mechanism of action is to activate the
immune system against melanoma. Imlygic’s Phase 3 data
was strong, with improved overall survival, disease control
rate, objective response rate, and time to complete response.
16.9% of Imlygic patients, and only 0.7% of control GM- CSF
alone patients, achieved CR. Imlygic is being evaluated in a
number of early- stage clinical trials across a number of indications including pancreatic cancer, soft- tissue sarcoma, and
head and neck squamous- cell carcinoma.
Genetic immunotherapy
A number of potential approaches using gene therapy for
hematological malignancies can be considered (Table22.3).
Proof- of- concept for immunotherapy as a valid approach to
the treatment of hematological malignancy has been provided by earlier clinical studies that demonstrated that infusion of allogeneic T cells can eradicate minimal disease in
patients relapsing after allogeneic transplant. Unfortunately,
these encouraging allogeneic responses require a haploidentical T- cell donor, which is not available to the vast majority
of patients. Another more widely applicable approach is the
use of autologous tumor cells that have been genetically engineered to express immunostimulatory cytokines. Trials
using this strategy in myeloma, low- grade lymphoma, leukemia, and chronic lymphocytic leukemia are being pursued.
Many tumor cells express unique antigens on their cell
surface, either alone or as proteolytically cleaved peptides in
association with MHC class I molecules. The specificity and
sheer quantity of these tumor- associated antigens (TAAs)
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Table22.3 Approaches togene therapy targeting hematological malignancies
Strategy Genes employed Problems and merits
Gene replacement p53, p16, Rb Gene delivery to every cell required
Specific to defective cancer cell
Gene inhibition BCR-
Suicide genes Thymidine kinase, cytosine deaminase Immunogenicity and bystander effect contribute
Drug resistance genes MDR- 1, DHFR, MGMT Stem cell gene delivery required
Immunotherapy IL- 2, IL- 12, B7- 1, CD40L, GM- CSF,
ABL, MYC, CCND1 (cyclin D1), BCL- 2 Gene delivery to every cell required
Systemic response, autoimmunity a theoretical
tumor- associated antigens (TAAs)
problem For TAAs, requires that tumors
express foreign antigen
may allow immune effector cells to distinguish tumor from
normal tissue (Figure22.3). Some of these TAAs have been
isolated and shown to be recognized by T-
cell receptor (TCR)
complexes on human cytotoxic T lymphocytes. This has
engendered gene therapy strategies to augment T- cellmediated eradication of tumors. Indeed, while to date cancer
has been the primary target of the bulk of clinical gene therapy protocols, this area has seen limited success. That is until
the results of a landmark study were published in 2006 by
Morgan and colleagues demonstrating definitive success in
this area. The cDNAs for the α and β chains of a TCR against
a melanoma antigen were subcloned into an oncoretroviral
vector and used to transduce peripheral blood lymphocytes
of melanoma patients. Following infusions of the transduced
cells into 17 patients, 2 patients demonstrated actual sustained tumor regressions as determined by standard criteria.
Furthermore, 1 year after infusions both of the responding
patients had high levels of gene- transduced cells in their circulation (20–70% of peripheral blood mononuclear cells).
A further evolution of this immune augmentation strategy
has led to the creation of chimeric antigen receptors (CARs).
As described above, Kymriah, the first gene therapy approved
in the United States is a CAR- based strategy. CARs use engineered artificial receptors that endow a patient’s own immune
effector cells with specificity against a TAA. A TCR specific
to the TAA is not required, rather, monoclonal antibodies
raised against the TAA are used as the platform for CAR
approaches. The single-
chain variable fragment of a monoclonal antibody against a TAA is fused to the intracellular
signal components of a TCR and other co- stimulatory signaling domains. These constructs can be transduced into the
patient’s own effector immune cells and, when re- introduced
into the patient, provide transduced cells with the ability to
recognize cancer cells expressing that TAA. CAR- T cell clinical trials have shown considerable promise treating blood
cancers. Clinical trials are underway for ALL, chronic lymphocytic leukemia (CLL), diffuse large B cell lymphoma
(DLBCL), follicular lymphoma, and multiple myeloma
among other indications. CD19, a B cell TAA, is the most
well-
studied CAR antigen. By 2015 over 200 patients had
undergone CD19- targeted CAR- modified T cell clinical trials for the treatment of ALL with reported positive response
rates hovering over 80%.
As more CAR-
T cell trials enter the clinic, variations of the
core strategy have emerged (as have playful adaptations of
the CAR acronym). Multiplexing TCR complexes to broaden
the immune response and overcoming antigen drift and subsequent tumor evasion, adaptation of recombinant LVs to
increase gene transfer frequencies, and incorporation of
methods to overcome immune dampening mechanisms
driven by the tumors themselves may increase the potency of
these strategies in future protocols. Examples of the new
CARs undergoing pre- clinical work are listed below. T cells
redirected for universal cytokine killing (TRUCKs) coexpress both a CAR and an anti- tumor cytokine. Cytokine
production can be induced by T cell activation and thereby
recruit immune cells to tumor sites. Universal CAR- T cells
are also engineered from allogenic T cells to eliminate endogenous TCR and/or HLA molecules and thus prevent graftvs- host disease (GVHD) and transplant rejection.
Self- driving CARs co- express both a CAR and a tumor
ligand chemokine receptor to enhancing tumor homing.
Armored CARs secrete active cytokines or express ligands
that improve CAR-
T cell efficacy and persistence. The
“armor” agent is chosen based on knowledge of the tumor
microenvironment to further enhance CAR- T cell efficacy
and persistence. Self- destructing CARs can be engineered by
transient CAR expression obtained by electroporation of
RNA into the cell or by inducing apoptosis with cell suicide
systems. Conditional CAR- T cells are inactive until binding
an exogenous small molecule that activates the CAR. Marked
CAR- T cells express both a CAR and a known tumor epitope
that existing therapies can clear. If adverse events result from
treatment, administration of the anti- epitope therapy will
clear the marked CAR- T cells and hopefully alleviate symptoms. Tandem CAR- T cells expresses a CAR consisting of
two linked recognition domains that both must interact with
a target on the tumor cell. A dual CAR- T cell expresses two
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336 Molecular Hematology
Antigen-presenting cell
gene of
Effect
t
T cell
https://t.me/med1917
Tumor cell
Gene-engineered tumor cells
Insert
interest
MHCI
MHCI
MHCI
γ interferon
Adhesion
Costimulation
IL–12
Adhesion
Adhesion
B7–1
Peptide
Peptide
Adhesion receptor
T-cell receptor
Costimulatory receptor
Adhesion receptor
T-cell receptor
Costimulatory receptor
Adhesion receptor
T-cell receptor
Costimulatory receptor
IL–2
Results in anergy
IL–2
T-cell recruitmen
and killing of
target cells
expressing
peptide
Signaling
defective
Restoration
of cell killing
IL–12
Figure22.3 Some facets of drug resistance immunotherapy. Efficient signaling of T cells occurs following adhesion, engagement of the
major histocompatibility complex (MHC) class I/T- cell receptor signal, and after a costimulatory signal is received. Under normal circumstances,
professional antigen- presenting cells (APCs) present processed peptides in the groove of the MHC class I complex to T cells. Tumor cells are often
deficient in one or all of the components required to function as APCs because they lack an appropriate tumor antigen, cannot process the
antigen, or are deficient in adhesion molecules, MHC class I or costimulatory molecules required to generate a T- cell response. These missing
components can be provided or expression enhanced using gene transfer techniques. By overcoming the deficiencies of the tumor cell, the
gene-
engineered cells can serve as autologous cancer vaccines presenting foreign antigen to the host T cells.
separate CARs with different targets; one CAR is fused to
only the CD3ζ intracellular domain, and the second CAR is
fused to only the costimulatory intracellular domain(s).
Thus, dual CAR- T cell activation also requires co- expression
of two targets on a tumor. A safety CAR contains both a CAR
and an antigen- binding domain linked to an intracellular
inhibitory domain. If the tumor expresses antigens for both
the CAR and the inhibitory domain scFv, the CAR will be
inactive. Thus, these CAR- T cells only become activated
when tumors display the CAR target but lack the inhibitory
receptor target. Bionic CARs combine CAR- T cells with
bispecific antibodies (antibodies featuring the benefits of
two monoclonal antibodies differing binding specificities in
one construct) to improve killing efficiency. Many of these
novel CAR- based systems may make their way into the clinic
soon. While the clinical data collected thus far is very encouraging, with five newly approved CAR- T therapies and
numerous CAR- T clinical trials launching only recently, a
considerably longer follow- up period will be required to provide data on long- term patient safety/efficacy responses.
Another facet of immunotherapy for cancer that has
received substantial interest is the application of gene
delivery techniques to induce specific immune responses
in patients by directing efficient antigen- presenting cells
(APCs) to present peptides from such TAAs to T cells. Where
patients have sufficient immune capacity for this kind of
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Molecular therapeutics inhematology: gene therapy 337
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manipulation, this can cause upregulation of the immune
response against that specific TAA. The most immunologically powerful (so- called professional) APCs are bone
marrow- derived dendritic cells. Dendritic cells express MHC
class I and II, B7- 1, B7- 2, CD40, ICAM- 1, and LFA- 3. They
are capable of presenting processed antigen for days, and are
potent stimulators of immunity when administered as vaccines to animals. Dendritic cells modulate immune responses
in part by secretion of IL- 12 (hence the notion of converting
tumor cells into APCs by introduction of IL- 12 and B7- 1
sequences). Dendritic cells can be readily expanded from
bone marrow progenitors in vitro using cytokinesupplemented medium (useful cytokines include Flt3L,
TNF- α, GM- CSF, IL- 4). Such cells may also be genetically
engineered by a variety of methods to express TAAs, thereby
presenting peptides in a proper context; this holds promise
for the immunotherapy of hematological malignancies and
for some solid tumors. Clinical trials have been performed in
this area targeting a number of TAAs. Results have indicated
that immune responses can be generated in patients. Current
work is focused on enhancing that immune outcome and
capitalizing on that facet to decrease tumor burdens and/or
treat metastatic disease.
As alluded to above, tumor cells known to express potentially antigenic peptides manage to evade host immunosurveillance and proliferate invivo. Thus, tumor cells may lack
or downmodulate expression of the necessary accessory signals required to induce expression by immune effector cells
of cytokines that are necessary for activation and directed
invivo expansion of cytotoxic T lymphocytes. The end result
is anergy, a failure of T cells to respond to the tumor antigen.
In addition to optimal presentation of antigen to the TCR,
efficient activation of naive T cells requires a second costimulatory signal. It is now appreciated that molecules of the B7
family (B7- 1/CD80, B7- 2/CD86) on APCs engaging CD28/
CTLA- 4 receptors on T cells play a key role in this process,
inducing autocrine IL- 2 production and T- cell proliferation.
Murine models have demonstrated that Trejection of tumors can be induced by transduction of tumor
cells with such costimulatory molecules. In the absence of
costimulatory signals, it is possible to bypass this requirement by ectopic expression of cytokines and thus overcome
or prevent anergy of the immune effector cells. Proof- ofprinciple that cytokine gene- transduced tumor cells can prevent tumor engraftment has been obtained. Such models
have also shown that transduction of the genes for various
cytokines, such as IL- 2, IL- 4, IL- 6, IL- 7, IL- 12, IFN- γ, GM-
CSF, and TNF- α, into murine tumors not only led to primary
rejection of the modified cells but often elicited protective
immunity against subsequent tumor challenge with unmodified tumor cells. Furthermore, in such models, synergy has
been demonstrated between molecules with varying mechanisms of action, for example IL- 2, IL- 12, and B7- 1.
cell- mediated
Methods toimprove gene therapy
safety andsuicide gene therapy
With the obvious importance of stably integrating vectors in
gene therapy protocols involving the hematopoietic system,
and given the deleterious outcomes in clinical trials for one
inherited disorder (see above), it is appropriate that the field
has directed attention to studying the safety of such gene
delivery agents. As discussed above, extensive sequencing
analyses have revealed that LVs and oncoretroviral vectors
have different integration patterns. Yet, in both cases, such
integrations are still fairly random. Efforts are underway to
tether the viral integrases to specific sequences in the
genome, thereby directing proviral integration into specific
areas of chromatin; however, to date, these strategies have
yielded limited success with the specificity of integration
being only minimally altered. Another strategy is the incorporation of insulator elements into the viral 3′ LTR. As the 3′
LTR of retroviral vectors is copied to the 5′ LTR during
theintracellular generation of provirus, insulator elements
provided in the 3′ LTR should yield inserted retrovirus
surrounded by insulator motifs, and thereby reduce the
likelihood of viral activation of host genes near the site of
insertion. A problem here is that such manipulations usually
reduce vector titer dramatically. Perhaps the most- studied
safety system is the use of gene transfer to endow target cells
of interest with “suicide” factors that allow their selective
eradication should deleterious outcomes arise. This approach
certainly has applications in hematological transplantation,
and also in numerous other developing research fields
employing different candidate populations such as embryonic stem cells and induced pluripotent stem cells.
At least three general applications of suicide gene therapy
can be envisioned. One is direct tumor therapy, where the
vector is injected into the tumor mass and patients are given
the prodrug, which is activated only in the tumor. The second application is in reduction of graft- versus- host disease
(GVHD) after donor lymphocyte infusion. Here, if symptoms of GVHD appear, productively transduced and transplanted cells can be selectively removed by addition of
prodrug. Lastly, if a truly portable system exists, such safety
elements could conceivably be incorporated into any cell
transplanted out of its normal context or into any gene
therapy vector in order to protect transduced cells from
genotoxicity and the development of leukemias should such
outcomes occur.
One class of suicide genes makes use of antibodyimmunotoxin conjugates engineered to specifically bind
unique cell surface markers transduced into the target cell
population. The antibody- immunotoxin conjugate is administered in vivo to specifically eradicate transduced transplanted cells. As this strategy relies on the administration of
toxic substances invivo a significant understanding of the
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338 Molecular Hematology
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expressing cells
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physiological response to the toxin is required to minimize
toxicity and maximize bioavailability of exogenous effector
molecules.
Another suicide strategy is the use of molecular switches
that engage apoptotic pathways in transduced cells. One
such strategy is the inducible caspase 9 (iCasp9) system. The
iCasp9 transgene encodes a chimeric receptor engineered to
be activated by the addition of an exogenous small molecule
referred to as a chemical inducer of dimerization (CID).
Induced dimerization of iCasp9 results in the activation of
the mitochondrial apoptosis pathway in transduced cells.
Clinically, the iCasp9 system has been used in five patients
who received stem cell transplantation for relapsed leukemia
and DLI. On addition of the CID, a rapid elimination of
transplanted T cells resulted. Thus, this promising suicide
system is currently being evaluated in several CAR- T- based
clinical trials. However, it is important to note that iCasp9based cell fate control requires transgene expression above a
certain threshold level to induce apoptosis, and thus transduced cells remaining post- CID application may prove
difficult to eliminate rendering this strategy less useful for
applications requiring a more complete elimination of
engineered cells.
Perhaps the best- studied suicide genes are enzymes
which, when expressed by transduced target cells, confer
susceptibility to drug- induced cell death by specifically converting normally non- toxic prodrugs into potent cytolytic
or cytostatic molecules. The gene most commonly employed
in clinical trials in this context is the human herpes simplex
virus (HSV) type 1 thymidine kinase transgene, which confers sensitivity to the drugs ganciclovir and aciclovir, among
others. The use of thymidine kinase is further enhanced for
some direct tumor applications by diffusion of the converted prodrug into neighboring cells and thus a bystander
effect occurs (Figure22.4). The obvious limitations of this
treatment approach are that not all cells targeted will be successfully gene modified and thus, even with the bystander
effect, only a fraction of malignant cells will be destroyed.
Nevertheless, applications of this type are in clinical trials
for the treatment of solid tumors. Another proven clinical
Vector carrying
herpes simplex thymidine
kinase (TK) gene
TK
protein
TK
protein
TK
Death of TK
Gap
junction
between
cells
TK
protein
Ganciclovir
prodrug
TK
TK
Figure22.4 Basis of suicide gene therapy. The metabolic product of the activity of the thymidine kinase suicide gene appears capable of
diffusing into neighboring cells via gap junctions, which join the cells together. This non- specific diffusion allows a greater effective cell- killing
percentage than might be predicted using direct gene transfer efficiency alone. Because cells are dying and releasing tumor antigen into the local
milieu of the tumor, it is possible that such suicide gene therapies will synergize with immune- based treatment strategies.
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application of this suicide approach, which may actually
have a higher probability of success, is in the prevention of
GVHD following transplantation. These trials were first
performed by Bonini and colleagues in patients who first
received T- cell- depleted allogeneic bone marrow transplantation followed by infusions of oncoretrovirally transduced
lymphocytes. The transduced cells demonstrated antitumor
activity in five patients. Three patients developed GVHD,
which was controlled by addition of ganciclovir.
The HSV thymidine kinase system relies on a slow and
fairly inefficient mechanism to convert ganciclovir into its
toxic form, and the enzyme itself is a foreign protein that has
resulted in anti- thymidine kinase immune responses being
reported in patients receiving transduced cells. This can lead
to premature clearing of the transplanted cell population. It
should also be noted that many transplant patients are
already on prophylactic ganciclovir to reduce the possibility
of cytomegalovirus infection.
For the above reasons, we have attempted to improve the
HSV thymidine kinase system. We implemented a human
enzyme (thymidylate kinase) that has been minimally modified to efficiently convert azidothymidine monophosphate to
azidothymidine diphosphate. This system allows for killing
of non- cycling cells through mitochondrial membrane disruption. In vitro and in vivo testing of such a suicide safety
system has been completed and recombinant LV that engineers expression of this variant enzyme is being produced for
eventual clinical trials.
Conclusions
the stage for a new era in human medicine. The field of
human molecular medicine will surely continue to expand
and make inroads in a therapeutically relevant way. It seems
likely that study of the blood system, both in the laboratory
and in patients, will continue to play a pivotal role in the further development of this field.
Further reading
Introduction
Brenner, M.K. (2001). Gene transfer and the treatment of hematological
malignancy. J. Intern. Med. 249: 345–358.
Jinek, M., Chylinski, K., Fonfara, I. etal. (2012). A programmable dual-
RNA–guided DNA endonuclease in adaptive bacterial immunity.
Science 337: 816–821.
June, C.H. (2007). Adoptive T cell therapy for cancer in the clinic.
J.Clin. Investig. 117: 1466–1476.
Melchiorri, D., Pani, L., Gasparini, P. etal. (2013). Regulatory evalua-
tion of Glybera in Europe– two committees, one mission. Nat. Rev.
Drug Discov. 12: 719–723.
Miller, A.D. (1992). Human gene therapy comes of age. Nature 357:
455–460.
Rosenberg, S.A., Aebersold, P., Cornetta, K. etal. (1990). Gene transfer
into humans: immunotherapy of patients with advanced melanoma,
using tumortransduction. N. Engl. J. Med. 323: 570–578.
Rosenberg, S.A. and Restifo, N.P. (2015). Adoptive cell transfer as per-
sonalized immunotherapy for human cancer. Science 348: 62–68.
Williams, D.A. and Smith, F.O. (2000). Progress in the use of gene trans-
fer methods to treat genetic blood diseases. Hum. Gene Ther. 11:
2059–2066.
infiltrating lymphocytes modified by retroviral gene
Some successes have been observed in clinical gene therapy
trials. Some concerns have also been raised. The development and implementation of gene delivery systems have also
led to the accumulation of important secondary knowledge.
For example, gene delivery methods are now routinely used
in most basic biology laboratories to study many different
processes. Viral entry mechanisms are now much better
understood and immune responses initiated following viral
infections are better characterized. As a result of the use of
integrating vectors, it has also now been demonstrated that
some T cells in humans are actually very long-
lived and that
multiple HSCs contribute to hematopoiesis. Much more has
also been learned concerning mechanisms that contribute
to the development of leukemias. Some concepts and
approaches to molecular therapy of diseases that interface
with the blood system have been described in this chapter;
many other therapeutic strategies are also being developed.
Indeed, the advent and success of human gene therapy trials
combined with rapid advances in gene transfer technology
and implementation of differential delivery systems, along
with the full sequencing of the human genome, are setting
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