Recombinant Moloney Murine Leukemia Virus

Most of the retroviral vectors that are used for gene therapy are based on the Moloney MLV. Vector replication is prevented by the deletion of the gag, pol, and env gene regions. The gag region encodes the capsid proteins; the pol region encodes reverse transcriptase and integrase; and the env region encodes proteins required for receptor recognition and envelope anchoring (Fig. 31-1). The genome includes long terminal repeats at either end that play a vital role in initiating DNA synthesis and regulating transcription of the viral genes. The gag, pol, and env gene products are supplied by a complementary packaging cell line. When a retroviral vector plasmid is introduced into a packaging cell line, viral RNA is produced, packaged into virions, and secreted into the medium. Each resultant viral particle is able to integrate itself into the genome of the host cell but is unable to produce additional viral particles because it lacks the gag, pol, and env genes. The transduced DNA sequences are stably integrated into the chromosomal DNA of the target T cells and in this way are transferred to cellular progeny of transduced cells. Highlights of results obtained to date with retroviral vectors include therapeutic studies in children with severe combined immune deficiency (SCID-X1), which will be discussed later in this review, as well as the gene-marking studies of Malcolm Brenner and others.^6,7 In the latter studies, it was shown that tumor cells within autologous stem cell transplant products could be responsible for tumor relapse, at least in patients with leukemia.

Recombinant Lentivirus

The most recently discovered members of the retrovirus family are the human and simian immunodeficiency viruses, which belong to a subclass of retroviruses known as lentiviruses.8,9 The development of HIV gene therapy vectors has several potential advantages based on the following characteristics:

Genetic modifications, such as the introduction of vesicular stomatitis virus (VSV) G protein into the lentiviral envelope, can widen the tropism of this vector. The first clinical study using a lentivirus vector was undertaken in HIV-infected patients.¹⁰ This study investigated the safety of infusing autologous T cells modified with an HIV-1–based lentiviral vector expressing an antisense gene against the HIV envelope. Five patients with HIV infections that were resistant to antiviral therapy and who had viral loads of >5000 copies/mL and CD4⁺ T-cell counts between 200-500 cells/mm³ were treated. The primary end points included adverse events, viral load, CD4⁺ counts, and the emergence of replication-competent lentivirus derived from the vector. In this phase I study, one subject was reported to have a sustained decrease in viral load. The CD4 counts remained steady or increased in the other four subjects, and sustained gene expression was observed. These preliminary studies support the safety of lentivirus vectors.

Hydrodynamic Gene Delivery

Hydrodynamic tail vein plasmid delivery results in high levels of transgene expression in the livers of rodents.⁷² Lower levels of transgene expression (100- to 1000-fold) are found in the spleen, heart, kidneys, and lungs. This simple nonviral gene transfer procedure entails the rapid delivery of naked plasmid DNA in a relatively large volume of physiological saline solution.⁵³ In a typical mouse weighing 20 g, the plasmid is delivered in a total volume of 2.0 mL over a period of 5 to 7 minutes.

Liposomes and Virosomes

In their most basic form, liposomes consist of two lipid species: a cationic amphiphile and a neutral phospholipid.71,73 Liposomes spontaneously bind to and condense DNA to form complexes that have a high affinity for the plasma membranes of cells, resulting in the uptake of liposomes to the cytoplasm by endocytosis. Many variations of this approach are used, resulting in varying levels of gene expression. Unfortunately, liposome-facilitated gene delivery is relatively ineffectual in vivo. More recently, some of the advantages of viral delivery vectors have been combined with the safety and “simplicity” of the liposome to produce fusogenic virosomes.⁷³ Virosomes are engineered by complexing the membrane fusion proteins with liposomes that have already encapsulated plasmid DNA. The inherent ability of the viral proteins in virosomes to fuse with cell membranes results in the efficient introduction of DNA to the target cell, providing improved gene expression. Viral vectors have limitations based on the size of transgene that can be incorporated; in contrast, no such limit exists for virosome or liposome technology (at least in theory).

Ballistic Delivery (Gene Gun)

The ballistic physical method of gene delivery uses microcarriers (usually gold particles) coated with DNA and “fired” using an explosive or gas-powered ballistic device called a “gene gun.”76–76 Once the particles are inside the target cell, the DNA is slowly released from the microcarriers, transcribed, and translated. This application has been used extensively in animal models, but its clinical use is restricted to exposable surfaces or ex vivo transduction because the particles do not penetrate tissues deeply.⁷⁷

Nanoparticles

Novel polymeric delivery systems (e.g., nanospheres) are potentially useful because the smaller the size of the condensed DNA particles, the better the in vivo diffusion to target T cells and trafficking within cells.80–80 Individual plasmid molecules can be condensed into a nanoparticle using detergents. Thus nanoparticle-based gene delivery targeting the neovasculature using an integrin-targeting ligand has been shown to result in tumor regression.⁸¹ Nonetheless, the size of the transgene that can be delivered is limited, and the primary focus has been on small interfering RNA (siRNA) delivery, which is discussed in a later section.

RNA Transduction

To date, the primary transgene source for DC transduction is DNA, although other antigen sources are also used with DCs, including peptides, recombinant or purified proteins, cellular extracts from tumor cells, apoptotic bodies, and RNA or DNA plasmid vectors. Nevertheless, the carrier of choice for loading DCs with tumor antigens is DNA or RNA.⁸⁴ Nucleic acid transfection leads to the display of multiple antigenic epitopes by both classes I and II major histocompatibility complex via the antigen-processing machinery of the patients’ DCs, resulting in the display of the “most appropriate” peptides. This strategy contrasts with vaccine strategies based on synthetic peptides, which require the knowledge of the patient’s unique peptide epitopes. Thus nucleic acid transfection of DCs offers several advantages for both immunologic and practical considerations.

The bias for the use of DNA vectors includes an increased stability compared with RNA, the ability to produce plasmids in large quantities, and the ease with which the sequence can be modified to regulate expression.⁸⁵ In several respects, however, RNA vectors are also advantageous when compared with DNA transfection.⁸⁵ RNA vector advantages include the ability to use total messenger RNA (mRNA) isolated from tumors to transfect DCs with no intervening cloning steps and the ability to express several or potentially all tumor-derived genes within DCs. Transfected RNA need only reach the cytoplasm of DCs, whereas DNA requires entry into the nucleus and subsequent transcription. Thus the low levels of antigenic epitope expression with RNA-transfected DCs could be advantageous, provided that expression levels are sufficient to generate a T-cell response.⁸⁴ The low levels of antigenic peptides presented by DCs allow only the T cells with high-affinity receptors to be activated, skewing the response toward T cells better able to recognize the tumor cells. Conversely, DCs presenting high levels of antigenic peptides allow T cells with low affinity receptors to be activated, thus masking or even preventing response of high-affinity T cells. This could result in T cells that kill cells with high antigen expression, but cannot kill tumor cells, which typically express low antigen levels. Thus RNA-transfected DCs potentially have greater efficacy for the activation of high-affinity T cells.⁸⁵

Oligonucleotides

Oligonucleotides are short single-stranded segments of DNA or RNA that, upon cellular internalization, can selectively inhibit the expression of a single protein.⁸⁶ Multiple forms of oligonucleotides are used in gene therapy, including antisense, siRNA, and ribozymes. Most of these constructs form a duplex with the mRNA or the pre-mRNA and inhibit their translation or processing, consequently inhibiting protein biosynthesis. This process occurs by multiple mechanisms, as discussed in the following sections.

Small Interfering RNA

RNA interference is a specific gene-silencing mechanism induced by the delivery of synthesized siRNA. siRNA is generated by dicer, an endonuclease that cleaves long double-stranded RNA molecules into fragments of 21 to 23 base pairs, which are highly specific for the nucleotide sequence of its target mRNA.87–90 These siRNAs associate with helicase and nuclease molecules and form a large complex, termed “RNA-induced silencing complex,” that unwinds siRNA and directs sequence-specific degradation of mRNA. RNA interference is a highly conserved molecular mechanism used by eukaryotic organisms to control gene expression and to defend their genomes against invaders, such as transposons or RNA viruses. siRNA-based technology has the potential to develop into an effective therapeutic strategy because it can modulate the expression of any protein, even undruggable target proteins such as transcription factors, with high specificity. However, the clinical development of siRNAs has been slow because of limited cellular uptake, low biological stability, and suboptimal pharmacokinetics. Indeed, naked siRNAs are easily degraded by nucleases and rapidly eliminated, and their negative charge and hydrophilic nature impairs cellular internalization.⁹¹ Advances have been achieved with lipid-based nanocarriers that are sterically stabilized by polyethylene glycol (PEG); however, PEGylated liposomes have technical challenges for siRNA delivery, and exchangeable PEG-derivatized lipids have been developed to improve tumor gene expression.⁹² Currently, lentiviral vectors provide a good option for siRNA delivery to cells, including nondividing cells.⁹³ Recently it has been shown that siRNA is active in vivo, resulting in therapeutic activity.⁹⁴ In one study, siRNA knockdown of the mutant K-ras oncogene had pronounced antitumor activity.⁹⁵ In this study, siRNA was delivered as a nonreplicative retroviral transgene and was shown to inhibit the relevant mutant K-ras and prevent anchor-independent growth and tumorigenicity. Despite these advances, RNA interference technology still requires refinement before its full potential can be utilized for routine clinical treatment.

Antitumor activities can also be obtained through siRNA knockdown of other critical components of tumor growth, metastasis, angiogenesis, and chemoresistance.⁹⁶ Stable transfection and expression of siRNA occurs with nonreplicating viruses^97,98; however, oncolytic virus vectors provide another method to extend bioactivity. This tumor-selective infectivity can restrict transgene expression to the cancer microenvironment, potentially reducing toxicity and extending transgene expression via viral replication and multiple cycles of infection within permissive cancer cells.^99,100 Furthermore, viral oncolysis can augment antitumor outcomes by siRNA-mediated therapeutic activity. In one study,¹⁰¹ the replication-competent oncolytic Adv ONYX-411 was used to deliver a mutant K-ras siRNA transgene, providing additive tumor growth–inhibitory responses. Therapy with ONYX alone or ONXY-411 with green fluorescent protein (GFP) siRNA as controls had significantly lower therapeutic activity.

Antisense

The principles of antisense technology are conceptually simple. Oligonucleotides are designed to hybridize to a defined target mRNA and to inhibit its translation into protein.102,103 This approach was first used in 1978 by Stephenson and Zamecnik¹⁰⁴ to inhibit the Rous sarcoma virus expression in chicken fibroblasts. Several antisense oligonucleotides are in clinical trials, and one has received FDA approval for the treatment of cytomegalovirus retinitis. Although it is relatively easy to synthesize phosphodiester oligonucleotides, they cannot be used as drugs because of their sensitivity to nuclease degradation. To improve their resistance to nuclease digestion, different chemical modifications are used, including phosphorothioates, methylphosphonates, and phosphoramidates.¹⁰⁵ These modifications increase the stability of oligonucleotides, but they also alter the capacity to hybridize with RNA and reduce cellular internalization.

Ribozymes

Ribozymes are RNA molecules capable of sequence-specific cleavage of mRNA molecules.¹⁰⁶ They selectively bind to target mRNAs, forming a duplex that is easily hydrolyzed, resulting in the suppression of specific genes.⁵⁰ Two types of ribozymes, the hammerhead and hairpin, have been studied extensively.¹⁰⁷ However, because the RNA backbone of ribozymes is an easy target for ribonucleases, they are biologically unstable in vivo⁵⁰ and thus are used primarily for gene suppression and for the induction of apoptosis and inhibition of proliferation.

Tissue-Specific Promoters

The production of proteins requires that the gene be transcribed into mRNA and then translated to protein,¹¹⁰ a process that is under multiple levels of control. The regulation of transcription is mediated by interactions between the enhancer/promoter region and the specific proteins or transcription factors that bind to this region. Activation or repression of promoters is achieved through interactions with transcription factors, such that in some tissues, specific proteins are expressed because the promoter for that gene is activated in that tissue alone. The success of transcriptional targeting is dependent on achieving differential gene expression in cancer cells compared with normal cells. Transcriptional control of gene therapy is an important goal for two reasons:

Criteria for selecting a promoter for use include consideration of the promoter’s strength, tissue specificity, and size. Promoter candidates include regulatory elements that are already expressed by the malignant cell, tissue-specific promoters, or externally inducible sequences. Unfortunately, many of these promoters lack sufficient activity, specificity, or both. To address promoter potency, promoters and enhancers that retain cell-specific function can be linked to transactivators. Additional strategies to enhance promoter activity in malignant tissues include the use of cell-cycle elements, normal or abnormal tissue differentiation factors, hormones, cytokines, chemicals, or physical stimuli. The classification of candidate promoters for cancer gene therapy (Table 31-1) includes tumor-associated, tissue-specific, and inducible promoters. These promoters are further discussed in the ensuing sections, as is the role of transcriptional regulation of replication-competent viruses, and specific examples are provided.

Tumor-Associated Promoters

Tumor-Specific Promoters

Conditional Replication and Inducible Promoters123,124

During evolution, various stress-response genes developed, and their promoters are now considered as gene therapy transcriptional regulation. Heat, hypoxia, glucose deprivation, irradiation, and chemotherapeutic agents upregulate stress response genes. Because of the relative weakness of tissue- and tumor-specific promoters, these inducible promoters are attractive as mediators of transient transgene activation. Promoters of these genes are also attractive for cancer gene therapy because they depend to a large extent on the biology of the tumor or are already induced by various therapeutic modalities.

Conditionally Replicative Viruses

Toxic or tumor suppressor gene expression from nonreplicative vectors, as a single therapeutic, is generally inadequate to control solid tumor growth.139,140 Thus replication-competent viruses have been developed as therapeutic agents for persons with cancer. Adv vectors are the most commonly used vectors in this context, although retrovirus, reovirus, HSV, and VSV are also used for the treatment of malignancies. The criteria governing the utility of replication-competent viruses include infection efficacy, replication selectivity, viral dispersion from the injection site, and evasion of the host immune response. Augmented gene transfer efficiency has been reported for Adv vectors based on the Coxsackie Adv receptor (CAR)–independent cellular entry pathways.¹⁴¹ Propagation of these vectors within tumor tissue remains a challenge, however. Recent improvements in our understanding of cancer biology have allowed the development of viral vectors with improved tumor-selective replication and restricted lytic effects to cancer cells. Dysregulation of the normal control over cell cycle and circumvention of physiological apoptotic signals can allow tumor-selective replication of an engineered virus and, subsequently, direct oncolysis by viral cell killing.¹⁴²

Conditionally Replicative Adenoviruses

Conditionally replicative Advs (CRAdvs) are designed by the deletion of Adv natural genes encoding cell-cycle regulatory proteins and/or by placing a tissue-specific promoter to control a viral gene essential for viral replication. An example of a vector with a deleted Adv gene is the deletion of CRAdv E1A (Table 31-2). This results in a loss of its conserved region 2, which precludes binding to the retinoblastoma gene and eliminates the inhibitory effect of the retinoblastoma gene on E2F. Consequently, the engineered Adv replicates selectively within cells in which the G1–S phase checkpoint is impaired (i.e., tumor cells).^143,144 Deletion of the Adv E1B 55 k protein was initially suggested to be selective for replication in p53-mutant cells, but this hypothesis has since been questioned.^145,146 Despite the mechanistic uncertainty, the E1B-deleted ONYX-015 virus selectively infects head and neck tumor cells, and clinical benefit has been suggested in patients with recurrent carcinomas.¹⁴⁷ Although ONYX-015 does not have a therapeutic transgene and relies on its lytic effect, it is the first CRAdv approved for cancer therapy. Shanghai Sunway Biotech Co. licensed world rights to ONYX-015 and obtained regulatory approval in China in December 2005.² This gene therapy product, H101 (ONYX-015), is a recombinant Adv modified to selectively replicate in and kill tumor cells with TP53 mutations. Clinical trials undertaken in China have been reported to show the safety of H101, with efficacy demonstrated in patients with HNSCC. Approval was based on a study that combined H101 and chemotherapy, which was reported to be effective in 78.8% of patients with this tumor.¹⁴⁸

Recently, mutants of human Adv 5 (Ad5) with enhanced oncolytic activity have been isolated using a procedure termed bioselection. In this approach, Ad5 was mutagenized and repeatedly passaged in a human colorectal cancer cell line. Mutants were found that replicate more rapidly than wt Ad5 and that lyse cells up to 1000-fold more efficiently.¹⁴⁹ Another strategy for designing CRAdvs uses tissue-specific promoters to drive expression of E1A, thereby restricting viral replication to specific tissues or tumors.¹⁵⁰ The application of heterologous promoters with Adv vectors is difficult because their activity and specificity are affected by viral enhancers and promoters. E1A gene expression under control of the alpha–fetal protein gene promoter induces relatively selective replication in hepatocellular carcinoma cells.¹⁵⁰ Control of Adv E1A expression by the PSA enhancer/promoter has been shown to confer prostate-specific oncolytic viral replication.¹⁵¹ Recently, a reengineered Adv vector with enhanced oncolytic efficacy that contained a novel regulatory circuit in which p53-dependent expression of an antagonistic E2F transcription factor was shown to inhibit viral replication in normal cells. In tumor cells, however, the combination of the p53 pathway defects and deregulated E2F allows replication at near-wt levels. This Adv vector also has demonstrated significantly enhanced efficacy for the treatment of human xenograft tumors compared with the extensively studied E1B-deleted Adv vectors.¹⁷

Another strategy that has been used employs a functional promoter/gene constellation–regulated Adv DNA replication, thereby providing selective transcriptional activation.¹⁵² These strategies to discriminate between tumor and normal tissue are based on selective DNA replication of Adv vectors with the entire E1 gene in tumor cells deleted. An E1 deletion is considered to abolish Adv replication; however, human tumor cell lines apparently can support DNA replication of Adv with an E1 deletion. Inverted repeats inserted into the E1 region of AdE1 vectors can mediate genomic rearrangements and bring a transgene into control of a promoter. Thus formation of a functional expression cassette depends on viral DNA replication, which is expected to occur specifically in tumor cells.

Structural Modification of the Fiber Protein

One approach to Adv retargeting involves engineering of the knob domain of the fiber protein. In this domain, the introduction of heterologous cell targeting peptides requires consideration of the structural limitations of the fiber three-dimensional configuration. The fiber is synthesized as a monomer, which undergoes trimerization before its attachment to the penton base. Thus any modification of the knob domain of the fiber must not impair trimer formation. In addition, the final quaternary configuration of the new fiber needs to make the incorporated ligand accessible to target cell receptor recognition and binding.

Recombinant Adv vectors have been constructed with a heparin/heparan sulfate-binding domain, consisting of polylysine residues added to the C-terminus of the fiber. Gene transfer to different mammalian cells has been obtained with a level of efficiency tenfold to 300-fold higher compared with unmodified vector.¹⁶⁴ The main drawback with this approach is the lack of specificity, because most mammalian cells express cellular receptors that contain heparin.

Modification of the Penton Base

Retargeting of Adv vectors has also focused on the modification of the penton base, which mediates the second step of Adv infection (i.e., internalization). Recombinant Adv vectors have been generated in which the RGD motif in the penton base has been replaced by the FLAG peptide. Complexing this vector with a bispecific Ab—consisting of a monoclonal Ab to the FLAG epitope and a monoclonal Ab to integrins—was shown to target T cells lacking the Adv fiber receptor, such as endothelial cells or human intestinal smooth muscle cells. Thus the first two steps of Adv infection binding and internalization are both mediated by A_v integrins.¹⁶⁵ In addition, recombinant Adv vectors can be constructed of chimeric penton base proteins that recognize tissue-specific integrin receptors.¹⁶⁶

Retroviral Vectors

Retroviral vectors were the first viral vectors to be targeted and the first to demonstrate the promise of vector targeting.30,108 Since that time, the challenge has been to incorporate targeting ligands without compromising vector entry into target T cells. Several approaches are now used to address this problem. One approach exploits pseudotyping, classically with the G-glycoprotein from the VSV, in which entry events are mediated through common membrane phospholipids.¹⁶⁷ Pseudotyped lentiviral and retroviral vectors have also been generated with glycoproteins from a variety of enveloped viruses, including Ebola virus, Marburg virus, rabies virus, lymphocytic choriomeningitis virus, Mokola virus, human foamy virus, gibbon ape leukemia virus, MLV, influenza virus, avian leukosis-sarcoma virus, and respiratory syncytial virus.^168–172 Although these pseudotyped vectors vary in terms of degree of envelope shedding, efficiency of packaging, titer, and stability, they can be concentrated to high titers for in vivo comparisons of cellular tropisms.

Another vector modification involves the ligation of polypeptides at the N-terminal of env to extend the host range of ecotropic MLV. Examples include erythropoietin, heregulin, and CD4.175–175 It should be stressed that coexpression of the wt env protein is necessary for infection to occur, possibly because incorporation of the engineered env protein in the virons can be facilitated by the oligomerization of both wt and chimeric env proteins. Another approach for engineering of the ecotropic MLV env protein involves the display of different polypeptide binding domains to the N-terminus of the ecotropic Moloney MLV surface protein. Examples include the N-terminal moiety of the amphotropic MLV env, single-chain Abs recognizing different cell surface receptors, heregulin, and epidermal growth factor. In some of these studies, infection specificity was redefined, although with lower efficiencies than those obtained with viruses expressing wt amphotropic envelopes.^176,177

Bifunctional bridging agents that recognize both the retrovirus and the targeted cell surface molecule provide evidence that retroviruses can enter cells via cell surface molecules that are not viral receptors. Such bridging agents have been used to infect human cells that are naturally resistant to ecotropic MLV-based vectors. The agents used usually consisted of an Ab to the MLV envelope protein connected to either another AB or a growth factor that would bind to the appropriate receptor such as the epidermal growth factor receptor, the insulin receptor, or major histocompatibility complex class I and class II molecules. Unfortunately, infection efficiencies for these agents are extremely low; emphasizing that binding of a retrovirus to a target other than the natural receptor cannot guarantee success.178,179

Nonviral Vectors

Nonviral vectors have no native receptor binding, but they do have a high incidence of nonspecific gene transfer from the high positive charge on many nonviral vectors.52–52 Many nonviral vectors transduce lung vasculature, potentially because of vector-mediated red blood cell aggregation and arrest in the lung subsequent to intravenous administration. The solution to the problem of nonspecific delivery is to shield the vectors, either with hydrophilic polymers such as PEG or with a ligand to reduce the surface charge. This type of shielding is applied successfully to lipid-based systems and to cationic polymer-based systems (polyplex).

Ligand-directed liposomes have shown some success in targeting tumors. Coupling of a synthetic AvB3-integrin ligand to cationic liposomes permits the selective delivery of a mutant RAF1 gene that causes apoptosis in angiogenic blood vessels within tumors. Systemic injection of the AvB3-targeted liposome results in apoptosis of tumor-associated endothelium and the regression of primary and metastatic tumor(s). This accomplishment highlights the extension of this approach from in vitro to in vivo efficacy.⁸¹

Another promising advance is to coat polyethylenimine-DNA polyplexes with a ligand, such as transferrin or transferrin plus PEG. Shielding by PEG or transferrin prevents nonspecific interactions with plasma proteins and erythrocytes but does not interfere with target cell interactions. When systemically administered in a subcutaneous tumor mouse model, the shielded complexes were shown to selectively transduce a well-vascularized, rapidly growing tumor. Although the specificity of this approach is high, the overall level of transduction is low.¹⁸⁰

In all the studies to date that use nonviral approaches, the need for relatively high dosing levels (~100 mg/mouse) suggest that innate clearance mechanisms might need to be saturated before substantial gene transduction can occur. Other potential issues that remain include determining whether the high doses used in the animal studies can be manufactured and delivered successfully for human studies. Attaining further improvements in the efficiency of gene transfer and better defining the toxicity profiles associated with these vectors are also critical steps.

Cellular/Targeted Vehicles for Gene Therapy

Tumor stroma are composed of multiple cell phenotypes including proliferating tumor cells, tumor fibroblasts, endothelial cells, myeloid cells, lymphocytes, and other cells. Some cells, primarily marrow progenitor cells, have a tumor-injury-tropism¹⁸¹ and can be used as vehicles for cancer gene therapy. These cells include mesenchymal stem cells, monocytes/macrophages, bone marrow stromal cells, and T cells with an ability to home to and infiltrate the tumor stroma^182,183 and can be used as cellular vehicles for gene delivery¹⁸³ or as targeted effector cells.^184,185 Pro-drug gene therapy delivered by mesenchymal stem cells is one of several treatment modalities under development that is providing an attractive tool for pro-drug delivery to the tumor mass and avoidance of systemic toxicity. Adoptive cell therapy with tumor-targeted T cells appears to be a promising approach to cancer therapy. In this strategy, a patient’s own T cells can be genetically modified to target antigens expressed on tumor cells through the introduction and expression of chimeric antigen receptors. These are fusion proteins consisting of single-chain variable fragments from monoclonal antibodies recognizing tumor-associated antigens and intracellular signaling domains, such as the CD3 z-chain cytoplasmic tail. When expressed by T cells, chimeric antigen receptors mediate redirected T-cell specificity to antigens expressed on the tumor, as well as to normal cells that express the targeted antigen. This approach has been translated into the clinical setting and patients with B-cell malignancies that express CD19 or CD20. Although cell-gene therapies are showing great clinical promise, several regulatory and clinical obstacles remain to be overcome and consensus still needs to be achieved regarding technical variables associated with cellular gene therapy.