STATE-OF-THE-ART HUMAN GENE THERAPY: PART II. GENE THERAPY STRATEGIES AND APPLICATIONS

Abstract

In Part I of this Review, we introduced recent advances in gene delivery technologies and explained how they have powered some of the current human gene therapy applications. In Part II, we expand the discussion on gene therapy applications, focusing on some of the most exciting clinical uses. To help readers to grasp the essence and to better organize the diverse applications, we categorize them under four gene therapy strategies: (1) gene replacement therapy for monogenic diseases, (2) gene addition for complex disorders and infectious diseases, (3) gene expression alteration targeting RNA, and (4) gene editing to introduce targeted changes in host genome. Human gene therapy started with the simple idea that replacing a faulty gene with a functional copy can cure a disease. It has been a long and bumpy road to finally translate this seemingly straightforward concept into reality. As many disease mechanisms unraveled, gene therapists have employed a gene addition strategy backed by a deep knowledge of what goes wrong in diseases and how to harness host cellular machinery to battle against diseases. Breakthroughs in other biotechnologies, such as RNA interference and genome editing by chimeric nucleases, have the potential to be integrated into gene therapy. Although clinical trials utilizing these new technologies are currently sparse, these innovations are expected to greatly broaden the scope of gene therapy in the near future.

1. Introduction

Human genetic disorders arise from mutations in the DNA genome, which in many cases abrogate the normal function of genes. To tackle these “loss-of-function” diseases, recombinant DNA technology fostered the hope that delivering the normal copy of a mutated gene to the patient will cure the disease. This “gene replacement” approach to treating single gene disorders represents the prototype of gene therapy. In addition to gene replacement, other gene manipulation strategies have been developed to meet the demand of treating mechanistically different diseases (Figure 1). For example, gene addition supplements therapeutic factors that are either protective or curative. Gene knockdown aims at diminishing gain-of-toxicity. Gene editing suits the need of introducing specific changes in the genome. Nowadays, gene therapy utilizes diverse gene delivery vehicles, comprises various gene manipulation strategies, and targets a broader range of human diseases such as infectious diseases. In Part I of this Review, we discussed the recent advances in gene delivery technologies, including using non-viral and viral vectors, in vivo and ex vivo routes of gene transfer. Here, we introduce various gene manipulation strategies employed in current human gene therapy applications, aiming to provide readers with a framework of comprehending the rapidly evolving field of translational gene therapy. For more comprehensive discussion focusing on specific diseases or approaches, readers are encouraged to explore the excellent review articles in References and research articles therein.

Figure 1.

Depiction of four gene therapy strategies. (A) A gene mutation (red dot) abrogates protein synthesis (red cross) and leads to disease. Gene replacement corrects the disease by providing a functional copy of the gene (green helix) and normal proteins (green circles). (B) Multiple genetic factors and/or environmental factors can result in a disease though a complex disease cascade (dashed arrow). Gene addition alleviates the disease by supplementing therapeutic genes that target a specific aspect of the disease mechanism. (C) A gene mutation leads to the production of toxic protein aggregate (clustered red circles) that causes a disease. Gene knockdown utilizes small RNAs (green combs) to inhibit the aberrant mRNA (black comb), thus preventing the synthesis of toxic protein aggregate and correcting the disease. (D) Gene editing by chimeric nucleases (circle and pie) is a versatile approach to make a targeted change from a disease-promoting sequence (red dot) to a disease-preventing sequence (green dot). Also see Figure 4.

2. Gene replacement therapy for monogenic diseases

Many human genetic diseases are clearly defined by a single gene defect, and gene replacement is a straightforward approach to treating these diseases. Not surprisingly, the majority of the most advanced clinical gene therapy development falls into the category of gene replacement, because the conceptually simple design provokes extensive investigation. In addition, animal models that mimic monogenic human diseases are more easily obtained by naturally occurring mutation or genetic manipulation, which provides a valuable platform for pre-clinical testing. Gene replacement can take place either directly in vivo or through ex vivo cell therapy.

Lipoprotein lipase deficiency (LPLD)

The first and only gene therapy product that has obtained marketing authorization in the Western world is Glybera, a recombinant AAV (rAAV) vector for treating a rare disease called LPLD by replacing with a functional LPL gene (Yla-Herttuala, 2012). LPL is an enzyme that breaks down fats, and patients suffering from LPLD are restricted to a low-fat diet and are prone to recurring, life threatening inflammation in pancreas (Gaudet et al., 2012). Glybera consists of a hyperactive copy of human LPL gene packaged in rAAV1 vector. Following one-time intramuscular injections at multiple sites, transduced muscle cells serve as a biofactory to continuously produce and secrete LPL into blood. The secreted LPL docks on the luminal surface of endothelial cells lining blood vessels, and helps to break down fats stored in circulating vesicles and to alleviate disease severity (Gaudet et al., 2013; Stroes et al., 2008). To protect the viral vector from host immune attack and clearance, immune suppression is recommended between three days prior to and twelve weeks after treatment (Ferreira et al., 2014a; Ferreira et al., 2014b). The success of Glybera is partially attributable to targeting an easily accessible and permissive organ, skeletal muscle, which has drawn considerable interest as a platform for gene therapy (Wang et al., 2014a).

Leber congenital amaurosis (LCA)

The eye also is considered as an ideal organ for gene therapy due to its small size, compartmentalized organization, slow cell turnover, and low risk of immunogenicity (Sahel & Roska, 2013). One form of LCA (a group of diseases with severe retinal dystrophy and blindness) is caused by loss-of-function mutations in the retinal pigment epithelium-specific protein 65kDa gene (RPE65). The RPE65 protein is an enzyme located in retinal pigment epithelium, and catalyzes biochemical conversion of molecules that are involved in phototransduction (Kiser & Palczewski, 2010). In a naturally occurring RPE65-deficient dog model, a single subretinal injection of rAAV-RPE65 restored retinal response and vision function, and the restoration was stable for 3 years (Petersen-Jones et al., 2012). The safety of a similar approach in human use was proved in three phase I clinical trials, and stable clinical benefits were documented for more than 30 treated patients (Bainbridge et al., 2008; Hauswirth et al., 2008; Jacobson et al., 2012; Maguire et al., 2008; Stein et al., 2011). Recently, readministration of rAAV-RPE65 to the contralateral eye was shown to be safe and effective in three patients (Bennett et al., 2012). However, the treatment does not prevent retinal degeneration, though it corrects retinal dysfunction, calling for a combination approach for long-term therapeutic efficacy (Cideciyan et al., 2013). Nevertheless, the RPE65 gene replacement trials opened avenues to gene therapy development for other monogenic eye diseases (Sahel & Roska, 2013).

Blood disorders

Hemophilia B is caused by the deficiency in the gene encoding blood coagulation factor IX (FIX). The FIX gene replacement programs that have been under extensive clinical evaluation aim to deliver rAAV-FIX to liver or muscle, so that the FIX synthesized therein can be secreted to bloodstream and function. Clinical efficacy has been encouraging, as treated patients show vector dose-dependent FIX production, and require less frequent or no infusion with recombinant FIX protein (High et al., 2014; Patel et al., 2014). An important finding from FIX replacement clinical trials is AAV capsid-induced cytotoxic T cell immune response that eliminates transduced cells and compromises therapeutic efficacy (Mingozzi et al., 2009). Although a concomitant immune suppression regimen has been shown to partially reduce the immune response and to improve efficacy, efforts are underway to better address this complication (Mingozzi & High, 2013). The clinical development of hemophilia B gene therapy provides valuable insight to future AAV-based gene therapies targeting liver or muscle as a biofactory to produce therapeutic proteins. Primary immunodeficiencies (PIDs) are a group of heterogeneous diseases characterized by an abnormal immune system. Many PIDs are monogenic, loss-of-function disorders that specifically impair hematopoietic stem cell (HSC) lineages that give rise to a variety of immune cells. The most severe form of PID is severe combined immunodeficiencies (SCIDs) that affect both T- and B-cells. Since the deleterious effects of genetic defects in PIDs are within HSC lineages, ex vivo HSC gene replacement is a rational therapeutic approach (Nienhuis, 2013; Zhang et al., 2013). Integrating viral vectors such as gamma-retroviral vectors and lentiviral vectors have been used to deliver genes into isolated HSCs, and to replace the adenosine deaminase (ADA) gene and the interleukin-2 receptor subunit gamma (IL-2RG) gene for ADA-SCID (Aiuti et al., 2009; Gaspar et al., 2011) and X-linked SCID (Hacein-Bey-Abina et al., 2010), respectively. Although the early clinical trials revealed the risk of insertional mutagenesis due to gamma-retroviral vector integration, lentiviral vector proves to be safer. Furthermore, integrating vector design has been refined and improved safety along with promising efficacy was demonstrated in recent preclinical studies (Carbonaro et al., 2014). The current experience with PID gene replacement has laid the foundation for developing other ex vivo gene therapies, which commonly utilize integrated gene transfer and require careful design and monitoring for genotoxicity.

Other promising gene replacement clinical trials involve treating respiratory diseases (such as cystic fibrosis and alpha 1 anti-trypsin deficiency) (Griesenbach & Alton, 2013; Mueller & Flotte, 2013) and leukodystrophies (degeneration of the white matter in the brain) (Biffi et al., 2011). Taken together, these human gene replacement applications have pioneered the gene therapy field. AAV vectors proved to be the most favorable for in vivo gene delivery due to high efficiency and low risks of immunogenicity and genotoxicity, whereas the most recent generations of integrating vectors with improved safety profile have shown encouraging results in ex vivo gene therapy.

3. Gene addition for complex disorders and infectious diseases

In contrast to a single gene defect underlying monogenic diseases, the combination effects of multiple genes and environmental factors cause complex disorders such as cancer and heart diseases, rendering gene replacement not feasible for these disorders. Complex disorders are often common, and represent the most urgent unmet medical needs. In addition to genetic disorders, infectious diseases also debilitate or kill a large population worldwide. Therefore, significant efforts of gene therapy development have been devoted to these diseases (Figure 2), which usually require a deep understanding of the disease mechanisms, and utilize gene addition to supplement a therapeutic agent that targets a common aspect of the disease.

Figure 2.

Breakdown of approved gene therapy clinical trials worldwide since 1989 according to disease applications. Data are from Wiley Gene Therapy Clinical Trials Worldwide (http://www.abedia.com/wiley/).

Heart failure

A common cellular defect in heart failure (poor heart function resulting in reduced blood flow insufficient to meet the body’s needs) is altered calcium transport in cardiac muscle cells, which is associated with reduced expression and activity of the calcium handling protein sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) not necessarily due to any defect in the corresponding genes (Kranias & Hajjar, 2012). Studies have shown that overexpression of SERCA2a in cardiac muscle cells improves cardiac function in various heart failure animal models (Sikkel et al., 2014). The Calcium Up-regulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID) gene therapy clinical trials aim to deliver the human SERCA2A gene to heart using a rAAV1 vector as a treatment for heart failure. In the CUPID 1 trial (phase 2a) involving 39 patients with advanced heart failure, a single intracoronary infusion of high dose rAAV1-SERCA2A improved cardiac function and reduced hospitalizations when examined at 12 months after treatment (Jessup et al., 2011; Zsebo et al., 2014). The phase 2b CUPID 2 trial involving 250 patients is underway to further evaluate the efficacy of high dose rAAV1-SERCA2A.

Cancer

The heterogeneity and refractoriness of cancer inspired gene therapists to develop innovative therapeutic approaches. Although many gene therapy attempts to treat cancer have been somewhat disappointing (Brenner et al., 2013), we highlight two gene addition strategies that have shown promising results, and are currently under clinical investigation. The first strategy involves a herpex simplex viral (HSV) vector with certain viral gene deletions, so that it only replicates in tumor cells and leads to oncolysis. Importantly, it delivers the gene encoding an immune stimulating cytokine known as granulocyte macrophage colony-stimulating factor (GM-CSF), which eventually helps immune effector cells to attack tumor cells (Goins et al., 2014). The critical role of GM-CSF gene addition in triggering a systemic anti-tumor immune response was demonstrated in a recent Phase III clinical trial treating melanoma, as intratumor injection of HSV^GM-CSF reduced the size of not only injected tumors, but also metastasized tumors (Andtbacka et al., 2014). The second strategy is to transfer a gene encoding a chimeric antigen receptor (CAR) recognizing CD19 (a B-cell surface marker) to ex vivo T cells, which are then reinfused to patients to treat B-cell malignancies. The T cells armed with anti-CD19 CAR were shown to reverse advanced-stage lymphoma in one patient (Kochenderfer et al., 2010). Currently, this strategy is being extensively evaluated in several independent clinical trials to treat B-cell malignancies (Kochenderfer & Rosenberg, 2013).

Infectious diseases

Using muscle-directed gene addition to provide antibody (also called vectored antibody) was recently exploited as an innovative approach to treating infectious diseases. In several pre-clinical animal studies, AAV vectors were used to deliver genes encoding antibodies against pathogen-derived antigens to muscle (Wang et al., 2014a). As a result, the secreted pathogen-specific antibodies in bloodstream continuously provide surveillance and clearance of future infectious pathogens such as HIV (Balazs et al., 2012; Johnson et al., 2009) and influenza viruses (Balazs et al., 2013). Clinical trials to treat HIV infection are at preparation phase with high expectation.

In addition, supplementing trophic factors is an actively pursued approach to treating cardiovascular diseases (Bradshaw & Baker, 2013) and neurodegenerative diseases (Simonato et al., 2013). It should be noted that gene addition should not be considered merely as a “Plan B” when gene replacement is not feasible. Rather, if proven to be successful in a certain application, the same gene addition approach is relatively easy and likely to be adopted for another similar disease condition, thus benefiting an even larger population of patients.

4. Gene expression alteration targeting RNA

RNA can be an intermediate (e.g. messenger RNA) or final (e.g. microRNA) gene product, with diverse functions in biology and disease. Due to the versatile roles of RNA molecules in controlling gene expression, gene therapy strategies specifically designed to target RNA or to produce effector RNA molecules deserve close attention. Here we briefly summarize two commonly utilized gene therapy strategies based on RNA biology.

Gene knockdown by RNA interference (RNAi)

In contrast to loss-of-function gene mutations that are often suitable to gene replacement, gain-of-toxicity mutations lead to the production of toxic gene products, and therefore require a gene knockdown strategy to diminish the toxicity (Figure 1C). RNAi is an RNA-mediated gene silencing process widely found in many eukaryotes including mammals (Deng et al., 2014). Since its discovery in 1998, RNAi has been extensively studied, and matured as one of the most powerful tools for gene silencing in both basic research and therapy development (Fellmann & Lowe, 2014) (Figure 1C). The effector small interfering RNA (siRNA, ~22 nt long, single-stranded) can be initially expressed in the form of short hairpin RNA (shRNA), followed by processing into siRNA to repress gene expression by triggering RNA degradation and/or inhibiting protein synthesis (Borel et al., 2014). Although the cellular toxicity associated with overexpressing shRNA is debatable, the design of artificial microRNA shuttle seems to be safer and can achieve acceptable gene knockdown efficacy (Boudreau et al., 2011; Boudreau et al., 2009). RNAi finds its use in treating diseases caused by toxic proteins derived from gain-of-toxicity gene mutations (San Sebastian et al., 2013), such as superoxide dismutase I in amyotrophic lateral sclerosis (Wang et al., 2014b). One challenge is that RNAi usually indiscriminately reduces the toxic mutant protein and the normal counterpart (the latter being expressed from the second gene copy in a diploid human genome), which can impair the gene’s normal function. In this case, allele-specific RNAi that targets only the mutant gene copy has been developed for diminishing the toxic effect of mutant huntingtin protein in Huntingtont’s disease, while preserving the normal gene copy’s function (Pfister & Zamore, 2009). RNAi has also been used to downregulate specific cellular pathways that promote complex diseases such as cancer (Deng et al., 2014). For diseases that are attributable to both loss-of-function and gain-of-toxicity, a dual vector can be designed to simultaneously knockdown toxic mutated gene product and replace with a normal gene, as exemplified in the gene therapy strategy for alpha 1 anti-trypsin deficiency (Mueller et al., 2012).

Reprogramming messenger RNA (mRNA) splicing by antisense oligonucleotides (AONs)

In mammals, most newly transcribed pre-mRNAs undergo splicing to form mature mRNAs composed of exons, which direct protein synthesis. Therefore, mRNA splicing regulates gene expression by inclusion or exclusion of specific pre-mRNA sequences for translation. In Duchene muscular dystrophy (DMD), many mutations in the DMD gene (encoding the dystrophin protein in muscle cells) disrupt reading frame and generate a premature stop codon in mature mRNA. Since the DMD gene is too big to be packaged in the most efficient in vivo gene delivery vector AAV for gene replacement, DNA or vector-derived RNA AONs (the latter being expressed using the U7 small nuclear RNA backbone) are designed to induce exon skipping, and to restore the correct reading frame, rather than replacing the entire DMD gene. (Goyenvalle et al., 2012a; Goyenvalle & Davies, 2011; Goyenvalle et al., 2012b) (Figure 3A). Although the resulting dystrophin protein is shorter than the normal version, it is partially functional and can significantly reduce disease severity. When designed properly, AONs can also induce exon inclusion. For example, spinal muscular atrophy (SMA) is caused by mutations in the survival of motor neuron 1 (SMN1) gene and loss of SMN protein. A similar gene, SMN2, differs from SMN1 by a single nucleotide change that disrupts a splicing enhancing signal and excludes exon 7 in most mature SMN2 mRNA molecules, thus resulting in no functional SMN protein production (Figure 3B). An AON was designed to force the inclusion of exon 7 in SMN2 mRNA, and therefore to restore SMN protein. This was achieved by designing the AON as a bi-functional molecule – binding to the target SMN2 mRNA and recruiting splicing factors that carry out desired splicing events (Hua et al., 2007; Madocsai et al., 2005; Mitrpant et al., 2013; Skordis et al., 2003) (Figure 3B).

Figure 3.

Reprogramming mRNA splicing by AONs. (A) Exon skipping to restore dystrophin in DMD. Normal splicing of DMD pre-mRNA leads to mature mRNA consisting of all exons that direct the synthesis of full-length dystrophin. In DMD, mutations in exon 50 result in abnormal splicing that excludes exon 50 in mature mRNA, which shifts reading frame and abrogates dystrophin production. AON binding blocks splicing signal in exon 51, which excludes exon 51 together with exon 50, thus restoring original reading frame and producing partially functional, albeit shortened, dystrophin. (B) Exon inclusion to restore SMN protein in SMA. Under normal condition, only the SMN1 gene produces SMN protein, whereas SMN2 gene is largely silenced due to a single nucleotide change in exon 7 (yellow bar), which excludes exon 7 in mature mRNA. In SMA, SMN1 is mutated (e.g. gene deletion), resulting in loss of SMN protein. A bi-functional AON binds to exon 7 of SMN2 pre-mRNA and recruits splicing factor (SF) that mediates the splicing pattern as in normal SMN1 pre-mRNA. Therefore, the silenced SMN2 gene is reactivated and provides therapeutic SMN proteins. Boxes: exons. Curved arrows: protein synthesis. Black stop signs: normal stop codons. Red stop sign: premature stop codon caused by frameshifting. Green stem loops: RNA AON embedded in modified U7 small nuclear RNA. DSB: double-strand break. DMD: Duchene muscular dystrophy. AON: antisense oligonucleotide. SMA: spinal muscular atrophy. SMN: survival of motor neuron.

It should be noted that we only focus on RNA effectors that are derived from corresponding gene transfer and transcription. Alternatively, synthetic DNA or RNA AONs can be delivered and serve similar functions in RNAi and modulating mRNA splicing. Such drug-like nucleic acid molecules are currently more widely used in clinical trials than gene transfer vector-derived RNA effectors (Deng et al., 2014). As gene delivery vector and vector design for expressing RNA are improving, we expect vector-derived RNA will play a greater role in human gene therapy.

5. Gene editing to introduce targeted changes in host genome

Target sequence-specific, designer nucleases have become a powerful tool kit for genome engineering. These chimeric nucleases consist of a sequence-specific and customizable DNA recognition module and a DNA cleavage domain (Gaj et al., 2013). To achieve sequence specificity, Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) utilize DNA-binding proteins (Joung & Sander, 2013; Palpant & Dudzinski, 2013), whereas the clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas system is based on RNA-guided DNA recognition (Mali et al., 2013). Regardless of the detailed mechanisms, these designer nucleases first induce double-strand breaks (DSBs) in targeted DNA sequence, and then realize a range of DNA modifications through two distinct DNA repair mechanisms, namely nonhomologous end joining (NHEJ) and homologous recombination (HR).

Gene disruption by NHEJ

A DSB can trigger NHEJ, a DNA repair mechanism that ligates the broken DNA fragments by introducing small insertions or deletions. This outcome is ideal for disrupting disease-promoting gene function (Figure 4A). For example, HIV infection of human T cells requires the presence of a T cell co-receptor called chemokine receptor 5 (CCR5). Utilizing ZFN to disrupt the CCR5 gene in T cells was shown to render the modified T cell resistant to HIV infection (Cannon & June, 2011). Several early phase clinical trials involving ex vivo CCR5 disruption for treating HIV infection are ongoing or completed (NCT01252641, NCT00842634, NCT01044654), with results being revealed in the near future.

Figure 4.

Gene editing by ZFN to introduce targeted changes in host genome. (A) Gene disruption through NHEJ. ZFN-induced DSB (white gap) in a disease-promoting gene (black box) is repaired through random insertion/deletion of a few nucleotides (red gap), which diminishes the disease-promoting function of the gene (red cross). (B) In the presence of a donor template with homology to the targeted genomic region, ZFN-induced DSB triggers homology-mediated recombination (dashed crosses), so that the therapeutic gene carried in the donor template is added to the host genome. If the DSB is targeted to a mutated gene region, HR can replace the faulty gene with a normal copy in the donor template. Black boxes: genes in host genome. Green boxes: therapeutic genes carried in donor templates. NHEJ: nonhomologous end joining. ZFN: zinc-finger nuclease. DSB: double-strand break. HR: homologous recombination.

Gene replacement and gene addition by HR

In the presence of a donor template consisting of similar sequences as the targeted DNA, a DSB can also trigger homology-mediated recombination, which can lead to either gene addition or gene replacement depending on where the DSB is targeted and the donor template design (Figure 4B). For example, in a hemophilia mouse model carrying a mutant human FIX gene, intraperitoneal injection of two AAV vectors expressing ZFN and a donor template, respectively, yielded addition of a partial FIX gene fragment harboring exons 2–8 to the mutant FIX transgene. The outcome was that the exon 1 of the mutant FIX transgene was fused with exons 2–8 from the donor template to reconstitute a full-length FIX open reading frame (Li et al., 2011).

The potential of genome editing in human gene therapy applications is evident, as a number of successful studies in human cells and model organisms have been reported (Li et al., 2014; Lisa Li et al., 2014). However, the specificity of editing needs to be rigorously tested to address possible off-target effects before widespread clinical use (Guilinger et al., 2014). In addition, editing efficiency needs to be further improved to meet therapeutic needs, particularly in in vivo gene therapy settings that require gene editing in an enormous number of cells throughout an organ or the whole body.

6. Conclusion

Among the current human gene therapy applications, gene replacement for treating monogenic diseases has been the most mature strategy, evidenced by successful marketing of Glybera and other late-stage clinical development. Gene addition benefits from the experience gained through gene replacement trials, and starts to show promising results in some of the most difficult-to-treat diseases after generations of vector refinement. In combination with some of the most exciting advancements in molecular biology, RNA-targeting gene expression modulation and DNA-targeting gene editing have opened new avenues for treating a much broader range of human diseases. We would like to emphasize that the classification of gene therapy strategies in this Review only aims to ease description and to help with understanding. In future gene therapy applications, the boundaries are expected to blur and cross, with continuous merging with novel biological discoveries and vectorology innovation.