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Gene Therapy Comes of Age: Review Summary
Gene Therapy Comes of Age: Review Summary
Retroviral vectors
MEDICINE The identification of a genome packaging signal
(11) and the creation of a producer cell line (12)
Gene therapy comes of age paved the way for design and facile production of
vectors capable of undergoing reverse transcrip-
tion and DNA integration but lacking replication
Cynthia E. Dunbar,1* Katherine A. High,2 J. Keith Joung,3 Donald B. Kohn,4 potential (13, 14). The g-retroviral vectors devel-
Keiya Ozawa,5 Michel Sadelain6* oped in the 1980s and early 1990s were the first
to be shown to deliver genes into repopulat-
After almost 30 years of promise tempered by setbacks, gene therapies are rapidly ing HSCs (15–17). C-type retroviruses were also
becoming a critical component of the therapeutic armamentarium for a variety of inherited adapted for efficient gene transfer into primary T
and acquired human diseases. Gene therapies for inherited immune disorders, hemophilia, lymphocytes (18–21). These vectors were used in
eye and neurodegenerative disorders, and lymphoid cancers recently progressed to first-generation clinical trials designed to deliver
approved drug status in the United States and Europe, or are anticipated to receive a normal copy of a specific defective gene into
approval in the near future. In this Review, we discuss milestones in the development of the genome of T cells or HSCs from patients with
gene therapies, focusing on direct in vivo administration of viral vectors and adoptive immunodeficiencies or cancer [reviewed in (22)]
transfer of genetically engineered T cells or hematopoietic stem cells. We also discuss (Fig. 1).
emerging genome editing technologies that should further advance the scope and efficacy Two other genera of the retroviruses were
G
ene therapies are bringing new treatment no clinical benefit or produce unexpected toxicities enabled gene transfer into nondividing cells but
options to multiple fields of medicine. that in some cases led to widely publicized patient still left quiescent G0 cells out of reach (25).
Forty-five years ago, Theodore Friedmann deaths (6). In 1996, a National Institutes of Health Lentiviral vectors can carry larger and more com-
provided a prophetic account of the poten- (NIH) advisory panel concluded that these dis- plex gene cassettes than g-retroviral vectors and
tial and challenges of using gene therapy appointing clinical results were due to insuffi- thus their development provided a critical ad-
to treat inherited monogenic disorders (1). Grow- cient knowledge of the biology of the viral vectors, vance for hemoglobinopathies (26). Lentiviral and
ing interest in gene therapy was inspired by the the target cells and tissues, and the diseases. The spumavirus vectors have another advantage over
recognition that—at least in principle—a single panel recommended that investigators return to g-retroviral vectors in that they preferentially
treatment might achieve durable, potentially cu- the laboratory and focus on the basic science un- integrate into the coding regions of genes. The
rative clinical benefit. Investigators hypothesized derlying gene therapy approaches (7). Develop- g-retroviral vectors, by contrast, can integrate
that in contrast to protein-based drugs that may ment of new vectors and a better understanding into the 5′-untranslated region of genes (27), a
require repeated infusion, gene-based therapies of target cells sparked a second generation of feature that increases the risk of potentially on-
delivered to long-lived cells might afford sus- clinical trials in the late 1990s and early 2000s. cogenic insertional mutagenesis in hematopoietic
tained production of endogenous proteins, such These trials produced evidence of sustained ge- cells (28). Lentiviral vectors are currently the
as clotting factors in hemophilia (2). Long-term netic modification of target tissues and, in some tools of choice for most HSC applications, but
cell replacement afforded by genetically engi- instances, evidence for clinical benefit. However, g-retroviral vectors are still used for certain ap-
neered hematopoietic stem cells (HSCs) may dura- progress was slowed by the emergence of serious plications in T cell engineering and HSC gene
bly alleviate a range of conditions, obviating, for toxicities related to high gene transfer efficiency; therapy (Table 1). Removal of endogenous strong
example, the need for lifelong enzyme adminis- for instance: insertional genotoxicity, immune de- enhancer elements from lentiviral and g-retroviral
tration or transfusion therapy (3, 4). Originally struction of genetically modified cells, and im- vectors using a “self-inactivating” SIN design (29)
envisioned as a treatment solely for inherited dis- mune reactions related to administration of certain is another approach that decreases the risk of
orders, gene therapy is now being applied to vectors (6, 8, 9). genotoxicity (30); this design is used in most cur-
acquired conditions, a concept best illustrated by Over the past 10 years, further maturation of rent clinical trials (Table 1). Integrating retroviral
genetic engineering of T cells for cancer immu- the “science” of gene therapy, safety modifications, vectors are reviewed in more detail in (31, 32).
notherapy. Recent clinical studies have found and improvements in gene transfer efficiency and
that single infusions of T cells engineered with delivery have finally resulted in substantial clinical Adeno-associated viral (AAV) vectors
synthetic genes encoding a chimeric antigen re- progress. Several gene and gene-modified cell- AAV vectors are engineered from a nonpatho-
ceptor can produce durable responses in a subset based therapies are already approved drugs, and genic, nonenveloped parvovirus that is naturally
of patients (5). over a dozen others have earned “breakthrough replication-defective. Wild-type AAV requires an-
Translation of gene therapy concepts to pa- therapy” designation by regulators in the United other virus such as an adenovirus or a herpesvirus
tient care began in the early 1990s but was plagued States and around the world. In this Review, we to replicate (33, 34). All viral coding sequences in
by repeated cycles of optimism followed by dis- highlight key developments in the gene therapy AAVs are replaced with a gene expression cassette
appointing clinical trial results. A number of these field that form the foundation for these recent of interest. One limitation of AAV vectors is that
early experimental therapies were found to provide successes and examine recent advances in targeted they cannot package more than ~5.0 kb of DNA
genome editing likely to transform gene therapies (in contrast to g-retroviral or lentiviral vectors, which
1
in the future. can accommodate up to 8 kb). AAV vectors are
Hematology Branch, National Heart, Lung and Blood Institute,
Bethesda, MD USA. 2Spark Therapeutics, Philadelphia, PA, USA.
predominantly nonintegrating; the transferred
3 Genetic engineering from viral vectors DNA is stabilized as an episome. This feature less-
Massachussetts General Hospital and Harvard Medical School,
Boston, MA, USA. 4David Geffen School of Medicine, University to genome editing ens risks related to integration but also limits
of California, Los Angeles, CA, USA. 5The Institute of Medical Recombinant, replication-defective viral vectors long-term expression from AAV vectors to long-
Science, The University of Tokyo, Tokyo, Japan. 6Memorial
Sloan Kettering Cancer Center, New York, NY, USA.
were the first molecular tool enabling efficient, lived postmitotic cells.
*Corresponding author. Email: dunbarc@nhlbi.nih.gov (C.E.D); nontoxic gene transfer into human somatic cells In the mid-1990s, two groups demonstrated
m-sadelain@ski.mskcc.org (M.S.) (10). Retroviruses and adeno-associated virus (AAV) long-term expression of a transgene following
in vivo muscle administration of AAV vectors to Nonhomologous end-joining (NHEJ)–mediated to cleave specific target DNA sequences, are
mice (35, 36). This seminal work led to the dem- repair results in the efficient creation of variable- now widely used for a myriad of applications in
onstration that AAV vectors could also efficiently length insertion or deletion mutations (indels) at basic research (56–58). A number of clever strat-
transduce a variety of target tissues in animal the site of the DSB, which generally inactivates egies that could eventually be applied clinically
models, including liver, retina, cardiac muscle, gene function. Homology-directed repair (HDR) involve the use of RNA-guided catalytically in-
and central nervous system, with specific tissue can be used to create specific sequence alter- active Cas9 (“dead Cas9” or dCas9) to turn genes
tropisms discovered for several naturally occurring ations in the presence of a homologous donor on and off by blocking transcriptional machinery
AAV serotypes and AAV engineered with opti- DNA template, which following recombination or recruiting epigenetic regulators (59, 60). Cor-
mized capsids (37). Improved manufacturing tech- results in correction of a mutation or insertion rection of mutations at a single-base level via Cas9-
niques [reviewed in (38)] increased both yield and of new sequences in a site-specific manner (44). based targeting of “base editors” has recently been
purity of AAV vector product, allowing proof-of- Early genome editing studies relied on engi- reported (61, 62).
concept studies in large-animal models of disease neering of specific zinc finger nucleases (ZFNs) Genome editing approaches offer a precise
(Fig. 2). Pioneering AAV gene therapy clinical (45) or meganucleases (46) for each individual scalpel for correcting or altering the genome
trials for hemophilia B were initiated in the late DNA target site to induce the required DSBs. and can overcome many of the drawbacks of
1990s, first testing delivery of AAV vectors to These nuclease platforms required specialized strategies that rely on viral vector–mediated
muscle via injection (39) and then moving to in- expertise to customize the DNA binding nucle- semi-random genomic insertion. For instance,
travenous administration, taking advantage of ase effector proteins for each cleavage target, genotoxicity due to ectopic activation of nearby
AAV2 liver tropism (40). These early trials estab- which limited their broader use and application. proto-oncogenes, knockout of tumor suppressor
lished safety but were limited by insufficient The demonstration in 2009 that the DNA binding genes, or perturbation of normal splicing should
dosing, and anti-AAV immune responses, most domain of bacterial proteins called transcription not occur with on-target editing. In addition, the
likely because many people carry neutralizing anti- activator–like effectors (TALEs) can be readily regulation of an introduced or corrected gene
Fig. 1. Historical overview of HSC gene therapy. HSCT: hematopoietic nuclease; TALEN: transcription activator–like effector nuclease; CRISPR/
stem cell transplantation; HSC: Hematopoietic stem cell; SCID: severe Cas9: clustered regularly interspaced short palindromic repeat (CRISPR)–
combined immunodeficiency; NHP: nonhuman primate; ZFN: zinc finger CRISPR-associated 9 (Cas9) nucleases.
Table 1. Clinical and product development landmarks for ex vivo gene therapies.
T cells Adult ALL* gRV CD19 (CD28) CAR-T (134, 143, 144) Memorial Sloan Kettering Cancer Center FDA 2014
....................................................................................................................................................................................................................................................................................................................
Pediatric ALL LV CD19 (4-1BB) CAR-T (145) University of Pennsylvania/Novartis FDA Oncology Advisory
Committee recommended
approval 2017; EMA 2016
.............................................................................................................................................................................................................................................................
gRV CD19 (CD28) CAR-T (146) National Cancer Institute/Kite
.............................................................................................................................................................................................................................................................
LV CD19 CAR-T, TALEN (74) Cellectis/Servier/Pfizer
knockout of TCR NCT02808442
and CD52
....................................................................................................................................................................................................................................................................................................................
Diffuse large B cell gRV CD19 (CD28) CAR-T (147) National Cancer Institute/Kite FDA 2014
lymphoma NCT00924326
.............................................................................................................................................................................................................................................................
gRV CD19 (CD28) CAR NCT02348216 Multiple academic sites/Kite FDA 2015; EMA 2016
.............................................................................................................................................................................................................................................................
LV CD19 (4-1BB) CAR-T (148) Multiple academic sites/Juno FDA 2016; EMA 2016
NCT02631044
.............................................................................................................................................................................................................................................................
LV CD19 (4-1BB) CAR-T NCT02445248 Multiple academic sites/Novartis FDA 2017
....................................................................................................................................................................................................................................................................................................................
*Abbreviations: FDA, U.S. Food and Drug Administration; EMA, European Medicines Agency; gRV, murine g-retrovirus; LV, lentivirus; ALL, acute lymphoblastic leukemia;
CLL, chronic lymphocytic leukemia; HSPC, hematopoietic stem and progenitor cells; X-SCID, X-linked severe combined immunodeficiency; ZFN, zinc finger nuclease;
BCMA, B cell maturation antigen; ARSA, arylsulfatase A; ABCD1, transporter gene mutated in adrenoleukodystrophy.
for CRISPR-based approaches; these components intravenous infusion. At least nine trials using Sciences, Engineering, and Medicine that brought
can be delivered by nonintegrating viral vectors or CRISPR-Cas nucleases have been approved by together an international group of scientists, cli-
transfected as mRNA or RNA-protein complexes regulatory agencies in China, primarily to knock- nicians, ethicists, patient advocates, and govern-
into target cells such as HSCs ex vivo. However, out PD1 expression in tumor-targeted T cells, and ment officials. This group published a report in
gene correction by HDR requires donor DNA, several have reportedly enrolled patients. 2017 laying out principles of governance and
which is more difficult to deliver, and HDR It is important to stress that in comparison to oversight for human genome editing, and pre-
appears to be particularly inefficient in certain standard gene transfer approaches, genome editing— senting a possible pathway for eventual use of
quiescent cell types such as long-term repopu- particularly that based on CRISPR-Cas nucleases— genome editing technologies to correct germline
lating HSCs (68, 69), although progress is being is in its translational and clinical infancy. A mutations for certain serious diseases (82). In the
made (70). number of potential feasibility and safety hurdles United States, federal government funds pres-
Genome editing as a therapeutic modality is exist that may affect clinical applications; these ently cannot be used for research on germline
rapidly advancing into the clinic (Table 1). En- will require further preclinical studies in appro- editing and clinical trials cannot be considered
gineered ZFNs have been used to disrupt CCR5 priate models and carefully designed clinical trials. for approval by the FDA. Similar restrictions exist
(C-C motif chemokine receptor type 5) expres- For example, the extent of “off-target” mutations, in many other countries. Clearly most countries
sion in human T cells (71) and HSCs (72) to ren- due to nuclease-mediated NHEJ or even HDR at are far from a societal consensus on germline
der these cells resistant to HIV infection. A phase alternative sites, is under intense investigation. editing. The acquisition of more efficacy and safety
I/II (73) study of T cell CCR5 editing has been Related questions under study are how best to data from studies of genome editing in somatic
completed, and a phase I trial of HSC editing is design nucleases or CRISPR gRNAs to avoid off- cells is critical before implementation of human
ongoing (NCT02500849). TALENs have been used target cutting and how to predict, screen for, and germline editing can be considered.
to make “off-the-shelf” third-party anti-CD19 detect on- versus off-target genome alterations
chimeric antigen receptor (CAR) T cells less likely before or during clinical applications (75). Nota- Gene delivery in vivo
Fig. 2. Historical overview of AAV gene therapy for hemophilia. AAV: adeno-associated viral vector; FVIII: factor VIII; FIX: factor IX; Mfg:
Manufacturing.
produced at therapeutic levels, but the expres- carry them, but other strategies will be required disorders are much more challenging targets than
sion persisted for only several weeks, in contrast going forward. Mendelian inherited disorders. Parkinson’s dis-
to the stable expression observed in preclinical ease (PD), which is characterized by loss of do-
studies of hemophilic dogs (85). Subsequent stu- The eye paminergic neurons in the substantia nigra and
dies revealed that expression of the factor IX Phase 1/2 clinical trials conducted by multiple a decrease in dopamine in the striatum, has
transgene in humans was of short duration be- groups have demonstrated improvement in visual been an intensely pursued target. Gene therapy–
cause of an immune response to the AAV capsid function following subretinal injection of AAV2 mediated transfer of dopamine-synthesizing en-
(42). In a later clinical trial that incorporated vectors expressing retinal pigment epithelium- zymes into striatal neurons has been found to
short-term immunosuppression (86, 87) (Table 2), specific 65 kDa protein (RPE65) in patients with normalize movement in a nonhuman primate PD
transgene expression persisted for years, resulting inherited blindness caused by mutations in the model (100). Early-phase clinical trials have estab-
in circulating factor IX levels that were 2 to 7% of RPE65 gene (92–94) (Table 2). A cohort of phase lished the safety of AAV vector–mediated gene
normal; this was sufficient to reduce bleeding and III–eligible subjects from one of the phase I/II delivery of aromatic L-amino acid decarboxylase
lessen the need for recombinant factor IX infu- trials continues to demonstrate clinical benefits (AADC), an enzyme that converts L-dopa to do-
sions. A subsequent trial involved the transfer of a lasting a minimum of 3 years after injection, with pamine; glutamic acid decarboxylase (GAD), an
transgene encoding factor IX Padua, a naturally observation ongoing (95); however, patients in the enzyme that modulates production of the neu-
variant of factor IX with high specific activity. The other two original trials have experienced regres- rotransmitter GABA (g-aminobutyric acid); and
transgene was carried by a vector containing an sion of visual function over similar follow-up periods neurturin, a neurotrophic factor (101–104). Promising
optimized AAV capsid and a liver-specific expres- (96, 97). At present, there is no clear explanation results were obtained with AADC gene therapy,
sion cassette. Patients showed a mean sustained for the differences in outcome because all used with additional early-phase clinical trials ongoing
factor IX activity level of more than 30%, resulting AAV2-based vectors. Subtle differences in manu- (Table 2). An early-phase trial of AAV2 vector
in complete cessation of factor IX infusions in 8 of facturing process, final formulation, design of ex- injection into the brain has also been conducted
Fig. 3. Historical overview of CAR-T cell therapy. CAR: chimeric antigen receptor; cGMP: current good manufacturing practices; DLI: donor leukocyte
infusion; LAK: lymphokine-activated killer; Mfg: Manufacturing; NK: natural killer; TIL: tumor-infiltrating lymphocytes.
(Table 2). This approach may be preferable to of-function mutations in the genes encoding vector production, ex vivo HSC manipulation, and
repeated intrathecal injections of an oligonucleo- interleukin-2 receptor g or adenosine deaminase pretransplant cytoreductive conditioning (116)
tide drug. (Table 1). Despite relatively low HSC transduc- all contributed to clinical benefit in several more
tion efficiencies with g-retroviral vectors, gene- recent trials. The metabolic disorder adrenoleu-
Ex vivo gene delivery via cell modified T-lineage cells were able to expand and kodystrophy and the lysosomal storage disorder
engineering: Monogenic blood disorders fill the empty T cell compartment, improving metachromatic leukodystrophy result in profound
and cancer immunotherapies immune function despite minimal levels of gene- neurologic degeneration and death in childhood.
Hematopoietic stem cells corrected cells in other lineages (108, 109). How- Lentiviral gene therapy clinical trials in both dis-
The clinical applications of gene therapies target- ever, several years after treatment, patients in the orders have been encouraging, with high-level
ing HSCs derive from the success of allogeneic X-SCID trials, as well as those for chronic gran- production of the missing enzymes from hema-
bone marrow transplantation for many genetic ulomatous disease and Wiskott-Aldrich syndrome, topoietic cells, including in the central nervous sys-
immunodeficiencies and blood cell diseases. These developed acute myeloid and lymphoid leukemias tem, and a slowing of neurodegeneration (117–119).
ex vivo approaches entail the transplantation of due to activation of proto-oncogenes adjacent to The clinical trial in adrenoleukodystrophy was
autologous stem cells in which an underlying proviral insertions, linked to strong enhancers the first reported using lentiviral vectors for HSC
genetic defect is alleviated or corrected [e.g., present in g-retroviral vectors and the propensity gene therapy.
adenosine deaminase deficit in severe combined of these vectors to insert near promoters (110–113). The hemoglobin disorders b-thalassemia and
immunodeficiency (SCID)], b-hemoglobin deficit These serious toxicities led to accelerated adop- sickle cell disease, which affect millions of patients
or structural alteration in hemoglobinopathies) tion of enhancer-deleted lentiviral or g-retroviral worldwide, have historically been an intense focus
(Table 1). Autologous transplants have an advan- vectors for HSC clinical gene therapies of immu- of gene therapy research, but require high effi-
tage over allogeneic transplants in that they do nodeficiency disorders. Encouraging clinical results ciency and substantial hemoglobin expression
not require a histocompatible donor, they avoid with these newer vectors have been reported in to correct the underlying pathophysiology (3).
Table 2. Clinical and product development landmarks for in vivo gene therapies.
FDA breakthrough/EMA*
Key publication(s) Institutional and/or
Cell type Disease Vector/transgene PRIME designation or
or clinicaltrials.gov no. industry partners
product approval
*Abbreviations: CNS, central nervous system; FDA, U.S. Food and Drug Administration; EMA, European Medicines Agency; AAV, adeno-associated virus; AADC, amino
acid decarboxylase; ZFNs, zinc finger nucleases; IDA, iduronate-2-sulfatase.
anemia via reactivation of endogenous fetal he- ical data bode well for CAR therapy of multiple inherited lipoprotein lipase deficiency), or are at
moglobin (HbF) expression. NHEJ-mediated dis- myeloma (136). risk of discontinuation of the program by the
ruption of the erythroid-specific enhancer element Current research aims to expand CAR ther- parent pharmaceutical company, as in the case of
responsible for expression of the BCL11A gene apy to myeloid malignancies and solid tumors Strimvelis, a g-retroviral vector HSC gene therapy
results in high-level HbF in animal models and (137, 138). These diseases present challenges be- treatment for adenosine deaminase–deficient SCID
in human sickle cell erythroid cells in vitro (122). cause reliable tumor-specific cell surface antigens (142). The U.S. Centers for Medicare and Medicaid
Similarly, disruption of a genomic locus to mimic have not yet been validated. In addition, there is Services announced a collaboration with the manu-
a genetic variant associated with hereditary per- a need for methodologies that facilitate CAR-T facturer of the first approved CAR therapy to
sistence of the fetal hemoglobin locus also shows cell entry into large tumors or immune-privileged provide the product under an “outcomes-based”
promise as a target for HSC genome editing to sites and that overcome tumor microenviron- approach, with payment collected only if patients
treat sickle cell anemia (123). ment signals that disarm T cells. Universal third- initially respond to the treatment.
party CAR-T cells that can be used “off the shelf” The past year has been marked by a flurry of
CAR therapy would allow more rapid and cheaper treatment scientific advances in genome editing, the publi-
Engineered T cells are emerging as powerful med- compared to autologous patient-specific T cells. cation of mature data from multiple clinical trials
icines for cancer (Fig. 3) (5). Chimeric antigen T cells lacking endogenous T cell receptors and/or demonstrating the efficacy and safety of gene the-
receptors (CARs) are synthetic engineered recep- major histocompatibility complex molecules to rapies for a wide variety of serious human diseases,
tors for antigen, which, in a single molecule, re- decrease the risk of GVHD and rejection are in and regulatory approvals of the first gene therapies
program the specificity, function, and metabolism preclinical or early clinical development as first in the United States. Scientists and clinicians
of T lymphocytes (124, 125). They consist of an steps toward this goal (62, 73). CAR-T cells have engaged in basic, translational, and clinical re-
antigen-binding domain, either from an immu- had a large impact on the treatment of certain search, supported by government and philan-
noglobin molecule or a T cell receptor, fused to cancers (139), and this success provides a founda- thropies, will continue to innovate and provide
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