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Molecular therapeutics inhematology: gene therapy 323
https://t.me/med1917
hemoglobin with mean total Hb levels increasing from <9 to
>11 g/dL by month 3 and maintained thereafter. All 31
patients treated with severe sickle cell disease characterized
by recurrent vaso- occlusive crises (VOCs) were free of VOCs
after treatment and through the duration of follow- up (2.0–
32.3months). Sickle cell disease patients had mean HbF of
approximately 40% of total Hb by month 4 post- treatment
and maintained thereafter. A regulatory submission was filed
with the EMA in late 2022 and with the FDA in early 2023.
With such considerable promise, CRISPR/Cas9 is considered
a breakthrough for the gene editing field, and in 2020, the
Royal Swedish Academy of Sciences awarded the Nobel Prize
in Chemistry to two pioneers of CRISPR technology, Jennifer
Doudna and Emmanuelle Charpentier. However, the usage of
such powerful editing technology raises ethical debates and is
not without controversy. In December 2019, He Jiankui, was
sentenced to 3 years in jail after using CRISPR to edit human
embryos leading to the birth of 3 babies while working at
Southern University of Science and Technology in Shenzhen,
China. While registered CRISPR/Cas- based trials are steadily
increasing in number, the great majority of gene therapy studies conducted to date have been gene augmentation protocols, involving co- expression or even amplified overexpression
of wild- type cDNA sequences.
Let us take a step back. An array of techniques has been
described to facilitate gene transfer into blood cells. A discussion of some of those methods is warranted. Such techniques can be broadly grouped as either physical methods or
viral vector methods. Physical methods of gene transfer are
generally of lower efficiency and usually provide only transient gene expression in the absence of selection. The advantages of physical methods of delivery are that genes are
transferred without viral sequences, which may affect the
biology of the target cell or the host. In addition, large or
multiple genes can be transferred, and gene transfer is independent of the proliferative status and cell- surface receptor
profile of the target cell. Those methods of physical gene
transfer that have been used in the clinical setting include
electroporation (electric fields that create channels in cell
membranes allowing passage of DNA into the cell), particle
bombardment (microscopic gold beads labeled with DNA,
for example, which are forced through the cell membrane by
CO
- driven pressure) and liposomal encapsulation of DNA
2
or RNA. Liposomal- mediated gene delivery has gained the
most acceptance in the clinical arena, driven by the higher
gene transfer efficiencies obtained with newer liposomal formulations and by safety considerations as an alternative to
viral vector- mediated gene delivery. In reality, the Pfizer and
Moderna vaccines developed for COVID- 19 are such deliverables: mRNAs coding for the SARS- Cov- 2 viral spike protein packaged in liposomal formulations, as is the Novavax
COVID- 19 Vaccine, polysorbate- 80nanoparticles delivering
engineered SARS- Cov- 2 viral spike protein itself.
Other non- viral gene delivery systems include the use of
plasmid DNA alone (so- called “naked” DNA), the use of
synthetic polymers, transposons, and the adaptation of bacterial gene delivery systems. The use of plasmid DNA alone
is particularly efficacious in specific tissues such as muscle.
Thus, nonviral DNA- based gene delivery is gaining in popularity for use in malignancy and infectious disease, and in
applications directed toward the vascular bed. In cancer,
DNA- based vaccines have been used in clinical trials where
the immunogenic antigen is encoded by the plasmid itself
with or without an added immunostimulatory gene. Yet,
while this approach is promising, clearly more work on modulating the biology of the immune response is needed.
Physical methods such as electroporation, direct DNA
transfer, exosomes, and liposome- mediated DNA transfer
have been used with varying levels of success for transferring
genes into hematopoietic cells. However, transgenes rarely
integrate with physical methods of transfer, except transposons, and the vectors are diluted in dividing daughter cells,
and thus are generally of little value to long- term stable gene
transfer applications. Nevertheless, when transient gene
expression is sufficient, such as in cancer immunotherapy
applications, and in gene editing where transient expression
of the “molecular scissors” responsible for editing can endow
a lifelong genetic change, physical methods of gene transfer
may find a niche.
Viral vectors forgene transfer
As an alternative to the physical methods described above,
protocols requiring higher efficiency gene transfer or stable
long- term gene expression make use of recombinant viral
vector transduction. The recombinant vectors used are modified from their wild- type state. The most commonly used
viral backbones in clinical gene therapy trials were previously based on murine oncoretroviruses and human adenoviruses; lately recombinant adenoand lentiviruses are preferred. The relative merits and disadvantages of some of these vector systems are outlined in
Table22.1. The particular nuances of the acquired or inherited disorder to be corrected determine which delivery system is more appropriate.
Viral gene transfer methods take advantage of facets of the
normal virus life cycle to facilitate the transfer of genetic
material into target cells. For the synthesis of recombinant
lentivirus- based gene transfer systems, for example, the
wild- type viral genome is modified by deletions of most
incumbent viral genes and insertion of therapeutic or marker
sequences in their place. The viral gene products necessary
to produce recombinant virions are then provided in trans by
transfections and replication- incompetent virions are produced. Replication- incompetent virions are deemed safe for
associated viruses (AAV)
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324 Molecular Hematology
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Table22.1 Some viral vectors used inthe application ofgene therapy
Vector Advantages Disadvantages Potential applications
Oncoretrovirus •
Adenovirus • Very efficient gene transfer
Helper- dependent
adenovirus
Adeno- associated
virus
Herpes virus •
Lentivirus • Designed to integrate
Pox viruses • Efficient gene transfer • Transient expression
clinical applications because they transduce target cells only
once; they are unable to cause a subsequent infection because
of the absence of secondary expression of viral genes required
for replication and virion packaging (Figure22.2).
Retrovirus- based vectors are attractive for the treatment of
conditions requiring stable long- term expression engendered
by genomic integration. Retroviruses also display a fairly wide
tropism, as well as minimal toxicity and minimal adverse
immune responses in human patients. Two retroviruses
commonly used in clinical gene therapy protocols targeting
hematopoietic cells are oncoretroviruses and lentiviruses.
Well- studied
• Integrates
• High transgene expression
• Large cloning capacity
• Not immunogenic
• Cell- cycle independent
• Designed to remain episoma
Very efficient gene transfer
• Cell- cycle independent
• Efficient gene transfer
• Large capacity
• Cell- cycle dependent
• Promoter silencing
• Safety issues
• Immunogenic
• Transient expression
• Pre- existing immunity
• Technically cumbersome
• Helper- virus contamination
• Transient expression
• Limited cloning capacity
• Helper- virus contamination
• Large- scale GMP production
• Can integrate with unintended
consequence
• Transient
• Cytotoxic
• Large- scale GMP production
• Public perception
• Can integrate with unintended
consequence
• Immunogenic
oncoretroviral vector include the packaging signal and the
long terminal repeats (LTRs), which are necessary for viral
integration and often drive transcription of the marking
ortherapeutic transgene in the absence of an added heterologous promoter. These deletions render the vectors
replication- incompetent while making approximately 6–8 kb
of space available for the insertion of desired transgene
sequences. Stable oncoretroviral packaging cell lines have
been engineered to minimize the chance of accidental
replication- competent retrovirus (RCR) production. Plasmids
used to create these stable packaging cell lines engineer
expression of key components of the parental virus but have
extensive deletions along with split genes and promoters.
Oncoretroviral gene transfer
These modifications have minimized recombination- prone
homologous sequences between the added gene transfer
Oncoretroviruses are double- stranded RNA viruses belonging to the gammaretrovirus genera of the family Retroviridae.
The viral RNA genome is reverse- transcribed into doublestranded DNA, which integrates into the genome of target
cells. Currently, oncoretroviral vectors derived from the
Moloney murine leukemia virus (MMLV) and other murine
oncoretroviruses are used for clinical gene transfer protocols
targeting hematopoietic cells.
For gene transfer vectors, the wild- type oncoretroviral
genome is modified by deleting most of the gag, and all pol
and env sequences. Viral sequences that are retained in the
vector encoding the transgene of interest and the stable
packaging cells, thereby significantly reducing the chance of
RCR being generated. Another measure of safety is naturally
present in oncoretroviral gene transfer systems as these
vectors, when packaged in murine- based packaging cell lines
at least, are rapidly inactivated by human serum. Taking a
lesson from commonly used lentivirus- based gene transfer
systems (see below), investigators have also generated recombinant oncoretroviruses with self- inactivating 3′ LTRs. Thus,
upon reverse transcription, this promoter unit is modified,
making it unable to drive transcription itself if productive
• Many involving hematopoietic cells
• Cancer immunotherapy
• Gene augmentation therapy
• Cancer immunotherapy
• Gene augmentation therapy via
muscle
• Brain delivery
• Cancer immunotherapy
• Pain management
• Cancer immunotherapy
• Many involving hematopoietic cells
• Nondividing targets
• Cancer immunotherapy
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Molecular therapeutics inhematology: gene therapy 325
Host DNA
Host DNA
https://t.me/med1917
Host nucleus
Infectious
HIV virus
Infectious
HIV virus
HIV genes
Infectious
HIV virus
HIV genes
Therapeutic
gene
Replication incompetent
lentiviral vector
Transfer
plasmid
Replication
incompetent
lentiviral vector
Packaging
plasmid
Envelope
plasmid
Target cell
Therapeutic
Therapeutic
transgene
Host nucleus
Figure22.2 Biology of the HIV retrovirus and the production of HIV- derived, replication- incompetent lentiviral vectors. For wild- type
retroviruses such as HIV, after binding and entry, the provirus integrates into the genome of infected cells. Viral proteins and new mRNA molecules
are produced. Virions are then assembled for the next round of infection. For HIV- derived recombinant replication- incompetent lentiviral vectors,
the gag, pol, and env genes have been removed to allow space for subcloning of the transgene of interest. To make a recombinant lentiviral
vector, the transfer plasmid containing the packaging signal and the therapeutic gene flanked by the HIV LTRs is introduced into a production cell
line along with separate plasmids engineering expression of gag, pol, and env genes in trans. The resulting replication- incompetent lentiviral
vector can then bind to, and be internalized by, a target cell wherein integration into the host genome occurs and the vector transgene product is
synthesized. Since the infected target cell lacks the genes necessary to form a new virion, the vector is not infectious secondarily.
recombination should occur and also making it unable to
drive transcription in the presence of viruses that may encode
cross- promoting functions. Lastly, along with the inherent
safety features described above, all viral supernatants and
patient cell samples that have been exposed to clinical- grade
oncoretroviral supernatants are extensively tested for the
presence of RCR and other possible contaminants prior to
infusion into patients.
Oncoretroviral vectors were one of the preferred gene
transfer vectors for early clinical gene therapy protocols and
are still used today. Their main advantage over other delivery
systems was stable integration of the vector into the host cell
transgene
No viral
proteins
No virions
released
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326 Molecular Hematology
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genome, allowing long- term transgene expression in the target cell and its progeny. These vectors also generated minimal immune responses themselves due to the extensive
deletions of wild- type coding subunits that they have undergone. Disadvantages of oncoretroviral vectors include the
requirement of target cells to be in cycle for genomic integration, the specificity of virion/receptor binding, which can
reduce infection rates into some target cells depending on
the env pseudotyping employed, the extensive periods of
ex vivo culturing that may be required to obtain efficient
gene transfer into key target cells such as human HSCs, and
the randomness of the integration event itself.
Some of the earliest work with oncoretroviral vectors
involved gene- marking studies in hematopoietic cells. By
marking transduced cells, the long- term distribution and
survival of transplanted cells could be followed invivo. The
reporter gene that was used most often in early clinical studies was the bacterial neomycin phosphotransferase (neoR)
gene which, when expressed, confers resistance to the neomycin analog G418. Other marker genes have also been used
and include those sequences encoding for the murine heatstable antigen, the human CD24 and CD25 antigens, the
truncated nerve growth factor receptor, modified CD4 or
CD19 or CD34 antigens, and an array of fluorescent proteins. The stable and unique integration pattern of proviral
DNA in the genome of marked cells can provide a permanent marker for individual hematopoietic or malignant cells
and their clonal descendants. This marking pattern can be
established using a polymerase chain reaction (PCR)- based
analysis that has been developed called ligation- mediated
PCR, which provides information on the actual site of integration of the provirus. Clinical applications in which gene
marking has provided new and important information
include the infusion of oncoretrovirally marked, autologous,
tumor- infiltrating lymphocytes into patients with advanced
melanoma and the infusion of oncoretrovirally marked bone
marrow or peripheral blood into patients with myeloid leukemia, myeloma, and neuroblastoma. The study of such
patients offers three important lines of investigation:
1
Is retroviral- mediated gene transfer relatively safe?
2 Do genetically altered bone marrow or blood stem cells
contribute to long- term hematopoiesis?
3 Do malignant cells or their precursors contribute to the
high relapse rates observed after myeloablative therapy and
autologous HSC transplantation?
A number of groups have reported the consequences of
infusing gene- marked bone marrow cells into humans and
the contribution of contaminating tumor cells in the graft to
disease recurrence. The first important observation from
these studies was that oncoretrovirus- mediated gene transfer
appears relatively safe. No detrimental effects, either on the
autograft or in patients, have been reported in these marking
studies. Replication- competent oncoretrovirus has also not
been detected at appreciable levels in patients participating
in clinical trials. That said, in two clinical gene therapy protocols that will be further described below, T-
cell leukemias
developed in five patients and appear to have resulted from
the integration of the therapeutic provirus into the regulatory region of a certain oncogene (see below). The second
important observation from earlier gene- marking studies
was that multiple HSCs contributed to long- term hematopoiesis, albeit at relatively low levels. Interestingly, rather
than all daughter hematopoietic cells being derived from a
single HSC, it thus appears that multiple stem cells contribute to the formation of the renewing blood system.
In the first gene- marking studies in children reported by
Brenner and colleagues, 2–15% of clonogenic hematopoietic
progenitor cells were marked after autologous bone marrow
transplantation. The marker gene was detectable for up to 4
years after transplant and was found in granulocytes, B- cells,
and T- cells, at least by PCR. In the earlier adult gene- marking
studies, however, oncoretroviral transduction of marrow or
peripheral blood HSCs has resulted in the detection of integrated vector in only a very low percentage of peripheral
blood cells. For example, in one study, although the marker
gene persisted for up to 2 years, neoR- positive cells could only
be detected intermittently with analyses employing PCR.
Work by a number of groups suggested that modification of
the transduction protocols would result in a significant
improvement in the engraftment of genetically modified
HSCs. Along these lines, since preclinical experience indicated that the use of bone marrow stroma enhances gene
transfer into HSCs, this approach was evaluated in some
early clinical trials. However, the results of this adaptation
proved to be no better than those discussed above and, in
general, are very similar to those observed in the pediatric
gene transfer studies.
In total, data on more than 40 patients enrolled in gene
marking studies during bone marrow transplantation for
acute myeloid leukemia, neuroblastoma, chronic myeloid
leukemia, breast cancer, myeloma, acute leukemia, or nonHodgkin lymphoma have been presented. Of the relapsed
patients, gene- marked tumor cells have been detected in a
high percentage in many studies. In one patient with acute
myeloid leukemia, the simultaneous detection of a cytogenetic marker along with the neoR gene confirmed that
gene- marked cells contributed to relapse. A second critical
observation was made a number of years ago when Rill and
colleagues reported that a multiplicity of neuroblastoma cells
in the graft contributed to relapse. The high frequency
ofgene- marked relapse, despite the very low frequency of
transfused malignant cells, strongly suggests that a large percentage of tumor cells in the graft contribute to relapse or,
alternatively, that tumor cells susceptible to retroviral gene
marking are uniquely capable of engraftment and clonal
expansion.
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Table22.2 Strategies foroptimization ofstable gene transfer into HSCs
Strategy Method
Inducing recipient cells to cycle Optimization of exvivo cytokine stimulation
Collection of cells during recovery phase after myeloablation or mobilization
Culture on stromal layers
Increased cell–virus contact Centrifugation of cells and virus during transduction (spinoculation)
Viral supernatant flowCoat dishes with fibronectin fragment
Higher viral titers and multiple exposures
Increase viral receptor levels on target cells Increase levels of amphotropic receptor by phosphate depletion
Transfer viral receptor into cell by adenovirus or adeno- associated virus
Target subpopulations of cells that have high levels of receptors
Alternatively pseudotyped recombinant retroviruses Exploiting GALV, RD114, 10A1 receptors for entry
VSV- G envelope to expand tropism and allow virion concentration
Modified retroviral vectors to increase efficiency and infect
non-
cycling cells
Positive selection of transduced cells Add positive selectable marker to vector: metabolic, fluorescent, cell-
Lentivirus-
and foamy virus- based vectors
through systems
surface
The gene- marking studies mentioned above set the stage
for the investigation of multiple maneuvers designed to
increase long- term gene transfer efficiency into HSCs. Some
of the strategies being pursued are described in Table22.2.
These include increasing the true direct target cell- to- virus
contact ratio by incorporating prior HSC enrichment, altering retroviral envelope utilization, or increasing colocalization of vector and cell in gene transfer protocols.
Newer vector systems improving on both oncoretroviral and
lentiviral backbones and incorporating alternative envelope
pseudotyping may also increase gene transfer efficiency,
while refinement of growth factor combinations may more
efficiently induce HSC cycling in shorter time periods, thus
improving retroviral integration without compromising
engraftment. Many of these approaches have now been
incorporated into clinical protocols and have likely contributed to some of the recent successes in this field as described
below.
Oncoretroviral vectors have also been used in chemoprotection studies. Cancer chemotherapy often involves induced
hematopoietic system toxicity leading to myelosuppression.
Gene therapy strategies aiming to protect hematopoietic cells
against the toxicity of chemotherapeutic drugs, i.e. chemoprotection studies, are under exploration. The gene marking
studies mentioned above suggest that normal bone marrow
cells could be removed, transduced with a vector engineering
expression of a drug resistance gene, and returned to patients.
Theoretically, such protected cells would then expand clonally after chemotherapy treatment and confer relative resistance to a cytotoxic or cytostatic agent, allowing further dose
escalation and a potential cure for some patients.
Furthermore, such drug resistance genes, if expressed in a
multi- cistronic format with a second therapeutic gene product, may even serve as selectable markers allowing exvivo or
in vivo enrichment or “preselection” of functionally genemarked cells, which may further enhance observed clinical
outcomes.
The original chemoprotection studies made use of the
human multidrug resistance 1 (MDR- 1) gene (P- glycoprotein).
The approach used for gene transfer there was generally
similar to that used for the human gene- marking trials.
MDR- 1 transduced and transplanted cells engrafted and
conferred drug resistance to bone marrow cells in vivo
andalso allowed for positive selection of MDR- 1 transduced
cells by chemotherapy. Infusion of oncoretroviral MDR- 1modified HSCs was found to be safe with no deleterious
effects of infusing such gene- modified cells observed, but
only minimal levels of gene marking were noted following
transplantation. Multiple studies have investigated an alternative strategy aiming to overexpress mutant methylguanine
methyltransferase (MGMT) in HSCs to allow them to survive
multiple rounds of chemotherapy targeting glioblastoma, for
example. The mutant MGMT was safe, allowed an increase
in the mean number of tolerated chemotherapeutic cycles,
and resulted in improved clinical outcome for the seven
patients treated.
The first human clinical gene transfer trials for inherited
single-
gene disorders used oncoretroviral vectors and
focused on adenosine deaminase (ADA) deficiency, a condition that leaves the body unable to produce leukocytes and
thus predisposed to infection. Recombinant oncoretroviruses containing the normal human ADA cDNA were transferred into either peripheral blood T lymphocytes, bone
marrow cells, or cord blood cells from ADA- deficient
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patients. T- lymphoid cells expressing the normal ADA gene
have a selective growth and survival advantage over ADAdeficient cells, even though patients were maintained on
pegylated- ADA enzyme replacement therapy (ERT) for ethical reasons. In those early studies, patients who had received
multiple infusions of autologous ADA- transduced blood
cells had increased levels of enzyme in their serum, and up to
20% of their peripheral blood T cells were found to carry
provirus for some period. Indeed, long- term follow- up has
revealed that one patient has maintained this level of marking for over 10 years. In two of the three initial trials in which
patients received autologous marrow or cord blood cells
transduced with ADA cDNA- containing oncoretroviruses,
between 12% and 40% of CFUs were transduced, and genetically marked cells were found for greater than 1 year after
infusion. In the one study, by Hoogerbrugge and colleagues,
in which provirally marked cells were not maintained for
greater than 6months, there was lower invitro gene transfer
efficiency of 5–12% CFU prior to transplant. In the cord
blood study, there was evidence for a selective growth advantage of T cells as there were higher levels of marked T cells
than myeloid cells even while the patients were maintained
on progressively lower doses of PEG- ADA therapy. ERT was
withdrawn from one patient and the number of T cells carrying the provirus increased to 30%; however, the total number
of B lymphocytes and natural killer cells dropped and the
patient had reduced immune function. That patient subsequently resumed PEG- ADA treatment.
Inherited immunodeficiencies, such as severe combined
immunodeficiency (SCID) of ADA (see above) and X- linked
SCID (see below), have become focal points for the potential
benefits and some of the potential hazards of gene therapy.
The above studies in ADA- SCID likely represent the first
tangible correction of an inherited disorder by stable transfer
of a therapeutic gene into primitive hematopoietic cells and
their progeny, although it is difficult to state this unequivocally due to the simultaneous administration of ERT.
However, those initial results have been surpassed recently
for ADA-
SCID in a study published by Aiuti and colleagues,
where productive transfer of the ADA gene was effected into
CD34+ cells of non- myeloablated recipients for whom ERT
was not available. Many of the incremental improvements in
the gene transfer protocols mentioned above were adapted in
that protocol and higher levels of gene marking (up to 25% of
CFU- C initially) and functional correction were demonstrated. Long- term multilineage hematopoietic cell marking
was also found in both patients. This study demonstrated,
without the confounding implications of ERT, that stable
long- term correction of this immune deficiency could be
accomplished using this therapeutic approach.
Impressive results have also been demonstrated in efforts
to correct another inherited immunodeficiency, X- linked
SCID. X- linked SCID (SCID- X1) is caused by a deficiency of
the common γ-
chain subunit of the receptors for the
cytokines interleukin (IL)- 2, IL- 4, IL- 7, IL- 9, and IL- 15 and
is thereby not a candidate for soluble factor augmentation
therapy. Expression of the γ- chain was also expected to offer
a growth advantage to productively transduced cells. This
was indeed the case and in a first report in 2000, two patients
were shown to have fully corrected immune function as a
result of the gene therapy. Since this landmark first description of this beneficial outcome, other X- linked SCID patients
have also been treated by this method. The original clinical
trials treated 20 patients and resulted in the majority of these
individuals retaining improved and stable immune function.
Yet even with these impressive results, this study highlights
areas where the field of gene therapy must still progress in its
understanding. This is because of reports that five of the
patients receiving the corrective γ- chain gene in two separate
clinical gene therapy protocols for SCID- X1 employing similar vectors and transduction protocols went on to develop
acute T- cell lymphoblastic leukemia- like disease approximately 3 years after the transplantation of oncoretrovirally
transduced cells. In at least two of these patients, the development of this proliferative disorder has debatably been
ascribed to a deleterious integration event that may have
caused the dysregulated expression of a proto- oncogene
called LMO2. However, it should be emphasized that since
analogous vector backbones and transduction conditions
have been used for other studies, such as the ADA- SCID trials mentioned above without the emergence of adverse
events, the possibility exists that these leukemia- like diseases
are a specific consequence of overexpression of the γ- chain
gene itself, since it impacts many diverse signaling pathways
that affect a number of cellular functions invivo. Still, a second attempt at SCID- XI gene therapy was launched in
2008with results published in 2012 detailing follow- up on
the nine patients enrolled at sites in France and the United
States. The oncoretroviral vector was modified to be a SIN
vector and to contain deletions in the viral enhancer
sequences that may have activated LMO2. Of the eight
patients that received treatment, seven displayed immune
system reconstitution, and analysis of vector insertion displayed significantly reduced clustering within LMO2 or
other proto-
oncogenes. Although very promising, these children will be followed for 15 years and longer to further assess
the efficacy and safety following treatment.
Results from another study involving the hematopoietic
system have also generated close scrutiny. Clonal dominance of cells with specific integration events was observed
in a recombinant oncoretrovirus- based clinical gene therapy trial for the X- linked form of chronic granulomatous
disease. These patients have impaired immune function and
cannot sufficiently resist bacterial and fungal infections due
to an inability of neutrophils to generate superoxide ions;
expression of the CYBB gene can abrogate this. Interestingly,
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the amplified integration locus involved the MDS1 and
EVI1 genes in that case, which differs from that mentioned
in the SCID- X1 trials above and may reflect alterations in
the activity of myeloid cells post- transduction with this
cDNA rather than a specific sequence preference for vector
integration.
Another particular group of inherited disorders currently
undergoing gene therapy pre- clinical and clinical trials are
the lysosomal storage disorders (LSDs). LSDs are genetic diseases that result from the loss of metabolic genes that normally work in the lysosome to break down particular cellular
molecules. Over time, the result of the disrupted enzyme
activity is an accumulation of substrate leading to a broad
spectrum of debilitating symptoms. As most LSDs are the
result of the loss or malfunction of a single enzyme, and as
even a small fraction of cells producing working copies of the
malfunctioning enzymes may be sufficient to cross- correct
lysosomes and positively impact the disease, LSDs are promising targets for gene therapy. Gaucher disease is an LSD
resulting from a deficiency in the enzyme glucocerebrosidase that is manifested mainly in macrophages, and it has
been proposed that this defect may be especially amenable to
treatment by therapeutic gene transfer into HSCs. Results
from two earlier clinical gene transfer studies targeting this
disorder have been reported. Mobilized peripheral blood or
marrow CD34+ cells from Gaucher patients were transduced
with an oncoretroviral vector that engineered expression of
glucocerebrosidase and then infused into non- myeloablated
autologous recipients. In both studies, transduced cells were
detected at low levels in blood and/or marrow leukocytes.
One patient who received cells transduced with an MFGbased oncoretroviral vector manifested increased levels of
enzyme activity corresponding to 50% of normal, which was
maintained for 12 months after infusion. No therapeutic
benefit or increased enzyme activity was detected in other
patients or in the other study. Development of viable murine
models for this LSD has been very beneficial for the testing
and implementation of novel gene therapy strategies involving some of the conditional manipulations mentioned above
or employing newer vectors. In addition to Gaucher disease,
gene therapy clinical trials are underway investigating a
number of other LSDs including Fabry disease, mucolipidosis, mucopolysaccharidosis type I, II, and III, metachromatic
leukodystrophy, neuronal ceroid lipofuscinoses, and Pompe
disease.Collectively, the results from the gene transfer studies for genetic diseases described above (and others not
directly mentioned) illustrate several points that will likely
impact on the clinical success of gene transfer protocols for
other single-
gene inherited disorders.
1 The presence in patients of cells carrying the provirus for
greater than 1 year has demonstrated the feasibility of gene
therapy and newer vector systems may further enhance
this.
The transfer of genes that provide a growth or survival
2
advantage can provide long- term expression and maintenance of transduced cells.
3 A selective advantage of transduced cells can compensate
for a modest gene transfer efficiency and yet, if too strong,
may lead to amplification of deleterious transformation
events.
4 Using the incrementally optimized protocols, gene delivery using recombinant viruses along with their subsequent
transgene expression levels may presently be sufficient to
correct a number of disorders that are directly or indirectly
manifested in the hematopoietic system.
Recently, Yescarta and Tecartus, two oncoretroviral
vector- based treatments, received FDA and EMA approval.
Both Yescarta and Tecartus are CAR- T- based therapies,
which we will explore in detail in a following section.
Yescarta and Tecartus both target CD19, treat a variety of
CD19- positive cancers, and are marketed by Kite Pharma.
Manufacturing differences account for the two differing
drug products. A study involving 359 patients with either
high- grade B- cell lymphoma or diffuse large B- cell lymphoma demonstrated that Yescarta is effective at prolonging
the time patients live without experiencing a disease event.
On average, patients treated with Yescarta experienced
event- free survival for 8months, as opposed to standard of
care’s 2- month average. Forty- one percent of patients given
Yescarta were event- free after 24 months of treatment, compared with 16% of patients receiving standard treatment. In
a study of 111 patients with diffuse large B- cell lymphoma
and primary mediastinal large B- cell lymphoma, Yescarta
improved outcome in many patients with 47% realizing a
complete response and 66% demonstrating a partial
response. In a study of 75 patients with follicular lymphoma,
91% responded to Yescarta, with 77% having a complete
response. Tecartus also demonstrated favorable outcomes,
with 59% of adult mantle cell lymphoma patients (44 out of
74) and 71% of ALL patients (39 out of 55) demonstrating a
complete response.
Although still used in groundbreaking clinical studies
such as the aforementioned CAR approaches, enthusiasm in
oncoretroviral vectors was somewhat reduced following the
leukemogenesis reports post-
X- SCID treatment described
above. We now know that oncoretroviral vectors have a predisposition to integrate at certain genetic “hot spots” including within genes, and in regions immediately surrounding
transcriptional start sites, leading to the potential for dysregulation of nearby endogenous genes. In an effort to overcome these obstacles and improve therapeutic safety and
efficiency, replication- incompetent complex retroviruses
based on lenti- retroviruses (lentiviruses, LVs), and human
foamy virus, have been developed. These vectors can integrate into these and other key non- dividing target cells and
are less predisposed to integration near active genes.
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Lentiviral gene transfer
LVs and human foamy virus are also members of the family
Retroviridae. They are sometimes termed “complex” retroviruses, in contrast to oncoretroviruses, which are termed “simple” retroviruses. Foamy viruses have a large genome (and
hence a large transgene- carrying capacity), do not cause any
known disease, and can be used for in vivo gene transfer
because they are not effectively inactivated by human serum.
LVs also show significant promise as gene transfer vehicles
and many laboratories around the world and subsequent clinical trials have adopted this alternative delivery system.
Current LVs contain less than 25% of the HIV- 1 genome and
seem to be much less susceptible to “shutdown” effects compared with recombinant oncoretroviruses. To further increase
safety, many laboratories now use third- generation recombinant LVs that have a self- inactivating (SIN) 3′ LTR. Much like
SIN oncoretroviruses, SIN LV minimizes the risk that
replication- competent lentivirus will be created. SIN vectors
also reduce the chance that the expression of endogenous
genes located near the insertion site will be enhanced by viral
LTR activity and reduce or eliminate transcriptional interference between the LTR and the promoter driving the expression of the transgene. To further minimize the chance of
recombination leading to the production of replicationcompetent lentivirus, some laboratories even use up to seven
separate plasmids in transient transfections of packaging cell
lines to generate recombinant virions (although three separate plasmids are more the norm). LVs can be readily pseudotyped with alternative envelope proteins, such as VSV- g,
which expands the tropism of the virions and also allows stable concentration of effective viral titer by ultracentrifugation. LVs can also transduce non- dividing cells. There is thus
considerable optimism that recombinant LVs will overcome
some of the shortcomings of MMLV- based oncoretroviruses
by facilitating delivery of transgenes of interest to a wide spectrum of cell types, including HSCs. Furthermore, due to a
marked increase in LVover previous methods, it may be possible to now better manage other secondary components of the exvivo gene therapy
procedure, such as enhancing engraftment of transduced cells
in recipients by minimizing culture periods and exposure to
differentiation- inducing cytokines, for example. Alterations
have also been made to LV backbones such as the addition of
a central polypurine tract and a woodchuck hepatitis virus
post- transcriptional regulatory element to possibly enhance
nuclear import or enhance transgene expression levels,
respectively.
As previously mentioned, LV genomic integration patterns differ from oncoretroviral integration patterns. Unlike
oncoretroviral vectors, which show a propensity to integrate
into promoter- proximal regions, LVs tend to distribute
throughout open chromatin and thus present less of an
mediated transduction efficiencies
insertional mutagenesis risk. That said, due to their increased
efficiency, LVs tend to integrate into permissive cells in
higher copy numbers than oncoretrovirus-
based vectors.
This increase can be good in order to generate higher levels
of transgene products for correction, but it also necessitates
careful planning concerning effective multiplicity of infections (MOIs) and the tolerance of the infected cell population to harboring multiple proviral copies. Indeed, it may
turn out that MOIs with LVs actually need to be reduced to
minimize this multiplicity effect in some protocols.
Importantly, in a landmark first LV study in HIV- AIDS
patients, no detectable recombination occurred between the
gene transfer vector and wild- type HIV- 1 even when the latter was present in very high copy numbers in patient blood.
Many gene therapy trials using LVs have been completed to
date and many more are underway.
As previously mentioned, Kymriah, the first gene therapy
approved in the United States, utilizes the LV delivery system.
Kymriah is a CAR-
T cell therapy based on infusion of autologous T cells transduced with an LV engineering expression of
a CD19- directed CAR for the treatment of ALL. Kymriah has
been evaluated in a number of clinical trials. One trial was
based on the treatment of 30 children and adults. Twentyseven patients experienced complete remission, including in
many patients whom stem- cell transplantation had failed.
The CAR- T cells were detectable in the blood, bone marrow,
and cerebrospinal fluid of patients who displayed a response.
All patients experienced cytokine- release syndrome, but this
was controllable with tocilizumab. Positive outcomes in a
subsequent global trial, ELIANA, helped secure FDA
approval. A phase II trial investigating Kymriah’s effect on diffuse large B- cell lymphoma has been completed (JULIET).
Kymriah has also been used in the clinic to treat chronic lymphocytic leukemia. In one study, 14 patients were treated with
autologous T cells transduced with Kymriah LV and infused
at varying doses. Greater than 50% of patients displayed a
positive response. The invivo expansion and persistence of
the CAR- T cells correlated with responses, no patient displaying a complete remission relapsed, and minimal residual disease was not detectable in patients displaying a complete
remission. All responding patients developed B cell aplasia
and experienced cytokine release syndrome.
Since Kymriah, several other CAR- T- based therapies have
gained regulatory approval. Breyanzi by Juno Therapeutics
was approved in February of 2021. It is also a CD- 19- based
CAR- T that targets certain types of non- Hodgkin lymphoma, including diffuse large B- cell lymphoma. Recently 2
CAR- T treatments targeting B- cell maturation antigen have
received approvals for the treatment for relapsed or refractory multiple myeloma. Abecma marketed by Bristol Myers
Squibb’s CelGene was approved in March of 2021, and
Carvykti marketed by Johnson & Johnson’s Legend Biotech
was approved in February of 2022. In a trial of 127 patients
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with relapsed refractory myeloma, 72% of patients partially
or completely (28%) responded to Abecma treatment.
Ninety- seven patients with relapsed or refractory multiple
myeloma experienced an overall response rate of 97.9% with
a median duration of response of 21.8 months following
Carvykti treatment.
Although the first LV- based gene therapy won FDA
approval in 2017, the first protocol in humans to employ
recombinant LVs was conducted in the early 2000s by Levine
and colleagues. That trial aimed to use the VRX496
(Lexgenleucel- T) LV to treat HIV infection. VRX496 is an
LV engineering expression of an antisense transgene against
the HIV envelope. The LV is delivered to autologous CD4 T
cells, which are then reinfused into the patient. Infection
with HIV results in antisense expression and disruption of
HIV replication. In one clinical study, a single IV infusion of
VRX496- containing CD4 T cells into five patients was found
to be safe. Engraftment was observed. No evidence of insertional mutagenesis was noted, nor was recombination
between the gene transfer vector and wild- type HIV. Patients
displayed improved CD4 counts, and in one patient, a significant decrease in viral load was seen. A following study
investigated multiple VRX496infusions into 17 patients and
noted a significant decrease in viral load. No evidence of
clonal selection of LV- transduced T cells or integration
enrichment near oncogenes was detected. Several other clinical trials have used gene therapy to target HIV. One study
involved treatment of four HIV patients with an LV delivering three RNAs: a shRNA targeting an exon shared by the
HIV genes tat and rev, an RNA hairpin transactivating region
decoy that antagonizes viral transactivation, and an antiCCR5- specific hammerhead ribozyme intended to block
viral entry. The therapy was well tolerated, and the shRNA
was detected in primary blood mononuclear cells and/or primary blood granulocytic cells at least 6 months posttreatment in all 4 patients. Another ongoing study is testing
an LV engineering expression of an anti- CCR5 shRNA and
C46 peptide to block HIV entrance into T cells.
LVs have also been used to treat X-
linked adrenoleukodystrophy (ALD), a severe brain demyelinating disorder
resulting from a deficiency in ALD protein. Autologous
HSCs were removed from two patients and modified with a
copy of the ABCD1 gene that expresses functional ALD protein by LV vector transduction. Between 24 and 30 months
post- treatment, polyclonal reconstitution had occurred, with
9–14% of granulocytes, monocytes, and T and B lymphocytes expressing the ALD protein. Beginning 14–16 months
after treatment, cerebral demyelination had stopped. Further
work led to the development of Skysona. Skysona gained
EMA approval in July 2021 and U.S. FDA approval in
September 2022. Slower progression to major functional disability (MFD) or death (MFD- free survival at 24 months post
symptom onset was 72% in Skysona patients as compared to
42% for untreated historical controls) was seen for early,
active CALD patients treated with Skysona.
Marina Cavazzana-
Calvo’s group has published on their
first patient treated with an LV delivering an anti- sickling β-
globin gene into autologous HSCs. Fifteen months after
treatment, therapeutic anti- sickling β- globin was still detectable and sickle crises, as well as other phenotypic hallmarks
of sickle cell disease, were absent in the patient. No adverse
events unrelated to busulfan conditioning were reported.
Cavazzana- Calvo’s group also used LVs to treat β- thalassemia,
a form of thalassemia resulting from reduced or absent
hemoglobin β chain synthesis that can result in severe anemia. More than 30 months after LV β- globin gene transfer, an
adult patient with severe β- thalassemia who had required
monthly transfusions since childhood, had been transfusion
independent for the previous 21 months. This work evolved
into Zynteglo, which gained EMA approval in June 2019 for
the treatment of the beta- thalassemia group of inherited
blood disorders, and the FDA’s approval in August 2022 for
the treatment of transfusion- dependent beta- thalassemia.
Several trials have been conducted using LV- based gene
therapy treatments for Wiskott–Aldrich syndrome, a rare
immunodeficiency associated with severe microthrombocytopenia, eczema, recurrent infections, and susceptibility to
autoimmunity and lymphomas. In one study, autologous
HSCs corrected by LV transduction were infused in seven
patients. Twenty- four months post- treatment six of the seven
patients were alive and showed sustained clinical benefit,
with substantially reduced disease- related days of hospitalization. The patient that died succumbed to a pre- existing
drug- resistant herpes virus infection. In a separate study,
three patients were treated analogously and showed stable
engraftment with improvements in platelet counts, immune
functions, and clinical scores. Importantly, in these three
patients, the LV- mediated therapy did not induce integrations near oncogenes, and no aberrant clonal expansion was
observed 20–32 months following the study. In a second
cohort of four patients treated by this same protocol, LVtransduced HSCs re- populated both the bone marrow and
the periphery, with a normal distribution of B- cell subsets,
and led to normal immunoglobulin and auto- antibody
expression in all treated patients. Conclusions from these
studies were that patients with high levels of transduced cell
engraftment achieved a normal platelet count and that sufficient immunological reconstitution does provide protection
from infection and autoimmunity in most patients.
Furthering the LSD clinical work presented above, Luigi
Naldini and Alessandra Biffi and colleagues have targeted
metachromatic leukodystrophy, a demyelinating lysosomal
storage disease caused by arylsulfatase A deficiency. Patients
received autologous HSCs transduced with a LV encoding
arylsulfatase A. Eighteen months post- treatment, the great
majority of patients displayed stable engraftment of
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gene- corrected cells, increased arylsulfatase A activity in circulating HSCs and in cerebrospinal fluid, a reduction of the
glycosphingolipid buildup normally associated with disease
pathology, and improved functional outcome. We also published interim results on an LV- mediated clinical trial for the
treatment of Fabry disease. In that study, five adult male
Fabry patients had HSCs collected, LV- transduced, and reinfused following minimal myeloablation to make hematological space. All patients have shown sustained vector
persistence and corrective enzyme production in plasma and
leukocytes (some over 5 years) to date. All patients have
demonstrated polyclonal hematopoiesis. Three patients have
come off of their enzyme replacement therapy (ERT) –
though all are eligible according to the study criteria.
Direct bilateral injection of an LV into the putamen was
used to restore dopamine production in patients with
advanced Parkinson’s disease. Fifteen patients received the
LV, three at a low dose, six at a mid dose, and six at a high
dose. During the first 12 months of follow- up, 51mild and
3moderate adverse events were reported; however, a significant improvement in mean motor scores off medication was
reported in all patients.
Attempts to use vectors other than traditional HIV- 1derived LVs have been made and are now entering the clinic.
One such strategy is based on equine infectious anemia virus
(EIAV) and has been used to treat neovascular age- related
macular degeneration (NVAMD). NVAMD is an eye disease
leading to vision loss. Endostatin and angiostatin were delivered by EIAV LV to patients with advanced NVAMD by subretinal injection in three dosing cohorts. Each dose was
well- tolerated with no toxicities. Long- term transgene
expression and reduced disease progression were noted.
The development of replication- competent lentivirus
(RCL) is a theoretical safety concern for individuals treated
by LV gene therapy. A recent study by Kenneth Cornetta,
Director of the National Gene Vector Biorepository, looked
at samples from 26 clinical trials including 460 transduced
cell products from 375 patients. All cell products were found
negative for RCL. Following infusion, 296 of the patients
were screened for RCL, and all were found negative. Across
all LV-
based treatments to date, no research subject has
shown evidence of RCL.
Adenoviral gene transfer
Recombinant adenoviral gene transfer systems are commonly used for some gene therapy applications, especially in
immunotherapy protocols. Adenoviruses infect cells efficiently invitro and invivo, express high levels of transgene
products, can infect both cycling and stationary cells, and
exhibit wide tissue tropisms. They can also be produced at
very high titers and have a large carrying capacity for foreign
cDNAs. Adenoviral vectors have been used to efficiently
transfer genes into a variety of human tumors by direct injection and into circulating hematopoietic progenitor and
malignant cells in ex vivo transduction procedures.
Nevertheless, circulating B and T lymphocytes have generally proven relatively resistant to adenoviral-
mediated gene
transfer. So have CD34+ hematopoietic cells until it was
found that serotype 35 fiber knob domains can help overcome this blockade. Furthermore, many patients have preexisting immunity to common adenoviral serotypes used in
clinical protocols. This can lead to a strong immune response
to the vector and severely restricts re- administration
efficacy.
The receptor for most adenoviruses used in gene transfer
applications has been identified as the coxsackie- andadenovirus receptor protein and has been useful in determining the mechanism of adenovirus binding and cell entry.
Adenoviral vectors do not integrate into the genome at an
appreciable frequency and are lost from most cycling target
cells within weeks of transduction. Thus, in comparison to
integrating vectors, adenovirus may appear comparatively
safe; however, they are not without their own set of risks. In
1999, teenager Jesse Gelsinger died due to a massive immune
response generated by the adenovirus vector he had been
treated with in a trial at the University of Pennsylvania.
Adenoviral constructs have since been refined with many
potent immuno- stimulatory viral sequences having been
eliminated so as to leave only the necessary inverted terminal
repeats and thus reduce the immunogenicity of the vector.
Oncolytic adenovirus vectors, which replicate only in permissive cancer cells, have been developed and hold promise
for the treatment of solid tumors and leukemic cells. For
example, one such vector system only replicates in cells harboring defective p53 tumor- suppressor gene function. With
these modifications, adenoviruses may be ideal vectors if the
generation of an immune response and/or short- term high
levels of transgene expression are desired (such as for vaccination); however, they are not suitable for therapies that
require long-
term expression or integration of the transgene.
Gaining European approval in 2021 and FDA approval in
2022, Adstiladrin, a treatment for non- muscle invasive bladder cancer, was the first approved adenoviral gene therapy.
The Ad5- serotype- based vector is administered by catheter
into the bladder once every 3months and targets the bladder
urothelium resulting in local production of a cytokine with
antitumor activity, interferon alfa- 2b. The pivotal Adstiladrin
study investigated 157 patients who received the vector by
intravesical instillation every 3months for up to 12 months
(dependent on drug tolerance and cancer recurrence). Fiftyone percent of treated patients experienced a complete
response with 46% of responding patients maintaining a
complete response for at least 1 year. It is presumed that low
dose and the local delivery of the adenoviral vector into a
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