Advances and Challenges in the Development of Gene Therapy Medicinal Products for Rare Diseases

INTRODUCTION

In 1972, Friedmann and Roblin anticipated that gene therapy might ameliorate human genetic disorders. ¹ Since then, several integrative and nonintegrative vectors have been generated. In parallel with this, hundreds of clinical trials have been initiated using ex vivo and in vivo gene therapy approaches with the aim of providing curative treatments for inherited and acquired diseases, many of which are life-threatening diseases affecting pediatric patients. ^2,3 In addition to approaches based on the use of viral vectors, the generation of new nonviral vectors and gene editing tools has facilitated the design of gene targeting approaches not only in preclinical models but also in human clinical trials. ^4,5

In this study, we present a historic view in the development of ex vivo and in vivo gene therapy approaches that have resulted in the approval of innovative gene therapy medicinal products.

PROGRESS OF EX VIVO GENE THERAPIES FOR MONOGENIC DISORDERS

Ex vivo gene therapy approaches are based on the collection of target cells from the body of affected patients, followed by their ex vivo genetic correction and re-infusion in the patient. Ex vivo gene therapy has been mainly used for the treatment of diseases that had been successfully treated by allogeneic cell transplantation. This is the case of diseases treated by hematopoietic stem cell transplantation (HSCT), such as primary immunodeficiencies, red blood cell diseases and other blood-cell related disorders. Additionally, several neurometabolic conditions have been treated by HSC gene therapy based on the potential for genetically corrected hematopoietic cells to migrate and release the therapeutic transgenic protein to cells of the central nervous system (CNS).

In all these HSC gene therapies, autologous bone marrow (BM) or mobilized peripheral blood (PB) CD34⁺ cells are purified, transduced ex vivo, and thereafter infused into the patient (Fig. 1), in many instances after conditioning regimens capable of depleting endogenous HSCs. ⁶

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Figure 1.

Schematic representation of ex vivo and in vivo gene therapies. The figure represents common procedures used for ex vivo and in vivo gene therapy. In the case of ex vivo gene therapies, autologous HSCs or keratinocytes are genetically modified ex vivo either with integration-competent vectors or with gene editing systems, and thereafter reinfused in the patient. In contrast to ex vivo gene therapy, in vivo gene therapy is based on the direct inoculation of vectors, in most instances AAVs, harboring the therapeutic sequences. Alternatively, nanoparticles carrying gene editing molecules are directly administered to the patient. AAV, adeno-associated viral; HSC, hematopoietic stem cell.

In addition to HSC gene therapy, ex vivo gene therapy has been also used for the treatment of different genodermatoses, a group of genetic diseases frequently associated with severe skin lesions. Similar to HSC gene therapy, autologous skin biopsies from affected patients are obtained to establish keratinocyte cultures, which are then genetically modified ex vivo and used to cover denuded areas of the skin (Fig. 1). ⁷

PIONEERING PRECLINICAL STUDIES OF HSC GENE THERAPY

The first preclinical studies demonstrating the insertion of genes into the genome of mouse HSCs were published in the years 1984–1986, and were based on the efficacy of gamma-retroviral vectors (RVs) to stably transfer marker genes into mouse BM cells. ^{8 –10} These experimental studies not only demonstrated the stable gene marking of self-renewing HSCs but also showed the possibility of tracking genetically marked HSCs characterized by different repopulations and differentiation properties.

Since RVs could only stably transduce dividing cells, and given that under physiological conditions most HSCs are quiescent, a new HIV-derived self-inactivating lentiviral vector (SIN-LV) system capable of transducing quiescent cells was developed by L. Naldini et al. in 1996. ¹¹ These SIN-LVs carry a large deletion in the U3 region of the 3′ LTR that completely inactivates its promoter/enhancer activity and in turn contain an internal promoter driving the transcription of the transgene. The relevance of these new vectors in HSC gene therapy became even more evident after the discovery of the genotoxic potential of RVs. Experimental studies conducted in 2002 at the Hannover Medical School showed for the first time the generation of leukemias in mice that had been infused with RV-transduced HSCs. Interestingly, these leukemias were associated with proviral insertions within the Evi1 (ecotropic viral integration site-1) proto-oncogene, whose transactivation by the viral LTR promoter was considered the primary leukemogenic event. ¹²

Subsequent studies related to these observations demonstrated that RVs had a preferential integration near the start of the transcriptional units, whereas LVs showed a preferential integration anywhere in the transcriptional unit. ¹³ Due to the different integration patterns of RVs and LVs and also because of the specific design of SIN-LVs, these vectors were shown to be much safer vectors compared with conventional RVs. ¹⁴ Similar to SIN-LVs, a second generation of RVs—SIN-RVs—was generated by C. Baum's team, which was also characterized by improved safety properties compared with the first generation RVs. ^{15 –17}

INITIAL HSC GENE THERAPIES IN PATIENTS WITH PRIMARY IMMUNODEFICIENCIES

In parallel with advances in the design and use of RVs and LVs in disease animal models, several HSC gene therapy trials were initiated in the 1990s. The first clinical trial showing at least a minimal therapeutic benefit was conducted at the NIH in two children with adenosine deaminase severe combined immunodeficiency (SCID-ADA). In this trial, autologous T cells were transduced with an RV that carried a wild-type copy of the ADA gene. In contrast to current HSC gene therapy protocols based on a single infusion of transduced HSCs, up to 12 infusions of transduced T cells were administered to these patients, who remained dependent on enzyme replacement therapy. ¹⁸

The efficacy of this gene therapy approach was improved by the combined administration of PB T cells with T cell-depleted BM cells, each transduced with a different ADA-RV. The cellular and humoral immunity of these patients was improved, and the discontinuation of the ADA replacement treatment resulted in progressive increases of corrected BM-derived PB T cells. ¹⁹ A further improvement of this protocol was based on the use of purified BM CD34⁺ cells and the administration of a cytotoxic nonmyeloablative conditioning to facilitate the engraftment of corrected HSCs. In addition, in contrast to previous trials, these patients had not been pretreated by any ADA-enzyme replacement therapy, thus enhancing the proliferation advantage of corrected T cells. A sustained hematopoietic repopulation with gene-corrected myeloid and lymphoid cells was observed in these patients, who also showed marked improvements in their immune system. ^20,21

Subsequent data from SCID-ADA trials demonstrated that RV-mediated gene therapy had a favorable safety profile and was effective in restoring the immune function of these patients in the long term. ^22,23 The clinical studies carried out by A. Aiuti et al. facilitated that in 2016 the San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET) and GlaxoSmithKline (TIGET/Telethon-GSK) obtained the European marketing authorization for Strimvelis, an advanced therapy medicinal product (ATMP) that consisted of SCID-ADA CD34⁺ cells transduced with the ADA-RV. These cells became the first genetically modified cell product approved as a commercial ATMP, which is currently licensed to Orchard Therapeutics (see Ref. ²⁴ and Table 1).

The results of a new clinical trial in two patients with a different primary immunodeficiency, SCID-X1, were reported by Cavazzana and Fischer in the year 2000 at the Necker Children's Hospital. BM CD34⁺ cells from these patients were transduced with an RV that carried the common γ-chain cytokine receptor (γc) gene and then infused into nonconditioned patients, as initially conducted in the first SCID-ADA trials. Normalized numbers of T, B, and NK cells with improved function were observed in most of these patients. ²⁵ Similar results were observed in a clinical trial conducted by A. Thrasher's team at Great Ormon Street Hospital (GOSH) in four patients, all of whom showed substantial immunological and clinical improvements. ²⁶

Strikingly, 3 years after the initiation of the SCID-X1 trial in France, two patients showed deregulated premalignant cell proliferation due to the RV enhancer trans-activation of the LMO2 gene oncogene. ^27,28 Similarly, clonal T cell acute lymphoblastic leukemias were observed in 2 of the 10 SCID-X1 patients treated in the United Kingdom, ²⁹ revealing for the first time the leukemogenic potential of RVs in humans.

During those years, another RV-mediated gene therapy trial was conducted by M. Grez in Frankfurt. In this case, two adults with X-linked chronic granulomatous disease (X-CGD) were infused with CD34⁺ cells that had been transduced with an RV carrying the gp91phox gene after conditioning with a nonmyeloablative treatment. In both patients, a high proportion of functionally corrected phagocytes were observed, although this process was associated with the expansion of gene-corrected myeloid cells due to insertional mutagenesis. ³⁰ Unfortunately, both subjects developed myelodysplasia and showed silencing of transgene expression due to methylation of the viral promoter. ³¹

Finally, another RV-mediated gene therapy trial was carried out at Muenchen University in patients with the Wiskott-Aldrich syndrome (WAS) who received busulfan conditioning before cell infusion. While 9 of 10 patients showed sustained engraftment and partial or complete resolution of the clinical signs of the disease (immunodeficiency, autoimmunity, and bleeding diathesis), 7 patients developed acute leukemia associated with RV genotoxicity. ^32,33

Risks associated with the use of RVs became evident in most HSC gene therapy trials, ³ which implied the necessity of improving the safety of therapeutic vectors in the clinic. In the particular case of SCID-ADA, an extremely low probability of insertional oncogenesis by the therapeutic RV has been observed, ³ probably due to the metabolic nature of the disease. Therefore, this is the only RV-mediated HSC gene therapy that continues under approved clinical practice.

SAFE AND EFFICIENT HSC THERAPIES WITH SELF-INACTIVATED RV AND LV IN MONOGENIC BLOOD CELL DISORDERS AND NEUROMETABOLIC DISEASES

The low genotoxic potential of SIN vectors was first demonstrated in preclinical studies, ^14,34,35 and thereafter in numerous clinical trials in patients with hematopoietic and neurometabolic diseases (see review in Ref. ³ ).

ADVANCES OF EX VIVO GENE THERAPY FOR RARE SKIN DISEASES

Following similar processes such as those developed for HSC gene therapy, ex vivo gene therapy has been also used for the treatment of patients with severe genetic skin lesions known as genodermatoses (Fig. 1).

The first ex vivo clinical gene therapy study in a patient affected by LAM5-β3-deficient junctional epidermolysis bullosa (JEB) was conducted by Mavilio et al. at the University of Modena and Reggio Emilia in 2006. Keratinocyte cultures from an adult JEB patient were transduced with an RV carrying the LAMB3 cDNA. Corrected epidermal grafts were then transplanted onto surgically prepared regions of the patient. A firmly adherent epidermis in the absence of blisters was observed in this patient during the 1-year follow up of this study. Analyses of the provirus integration sites showed that regenerated epidermis was maintained by a defined repertoire of transduced stem cells and did not evidence genotoxicity risks such as the ones reported in RV-mediated HSC gene therapy. ⁷⁶

In 2016, the results of a clinical trial in patients with recessive dystrophic epidermolysis bullosa, another devastating skin disease generated by mutations in collagen type VII alpha 1 chain (COL7A1), were reported by Siprashvili et al. from Stanford University. Autologous keratinocytes from four patients were transduced with an RV carrying full-length hCOL7A1 cDNA, and then transplanted onto each of the patients. This trial showed expression of type VII collagen at the dermal–epidermal junction. Although wound healing was noted in some of the corrected grafts, variable responses that generally declined over time were observed. ⁷⁷

In 2017, a compassionate life-saving gene therapy treatment was initiated at Ruhr University Hospital and the University of Modena for a patient with a devastating life-threatening form of JEB generated by a splice site mutation in LAMB3. A skin biopsy from a nonblistering area was used to establish keratinocyte cultures, which were then transduced with an RV carrying the LAMB3 cDNA. Transduced epidermal grafts were applied on prepared dermal wound beds. The results of this clinical study showed that transduced keratinocytes regenerated a fully functional epidermis on the patient. Insertion site and clonal tracing studies revealed that human epidermis was sustained by a limited number of long-lived stem cells that could extensively self-renew and repopulate terminally differentiated keratinocytes. ⁷⁸

A more recent trial was performed at the GOS Institute of Child Health in six patients with Netherton syndrome (NS) caused by mutations in SPINK5. In this trial, keratinocytes were transduced with an SIN-LV carrying the SPINK5 under the control of the human involucrin promoter. Genetically modified epithelial sheets were successfully generated in three subjects, although only transient functional correction was observed. ⁷⁹

The advantages and also limitations currently associated with the ex vivo gene therapy of patients with severe skin diseases have been recently described. ⁷ Alternative approaches based on in vivo gene therapy have been developed for the treatment of patients with dystrophic epidermolysis bullosa (DEB), in this case using a herpes simplex virus type I (HSV-I), which has very recently obtained marketing authorization in the United States (see Table 1).

THE (R)EVOLUTION OF IN VIVO GENE THERAPY

Efforts to deliver genes in vivo for the therapy of human diseases, whether inherited or acquired, began at the beginning of the 1990s around the same time ex vivo gene therapy was developed. In those times, the most efficient vector system that emerged for transducing several tissues and organs to a high level, particularly the liver, was based on a replication-defective adenovirus serotype 5. ⁸⁰ However, its short-lived transgene expression due to cell-mediated immunity against transduced cells that expressed residual adenoviral proteins made it clear that a “naked” vector was needed in vivo. ⁸⁰ In addition, acute toxicity was observed following systemic administration of Ad vectors which caused the first gene therapy death in a clinical trial. ⁸⁰ While “gutted” helper-dependent adenoviral vectors were generated that were devoid of any adenoviral coding sequences and resulted in long-term transgene expression, their systemic administration was still associated with acute toxicity. Therefore, a less immunogenic vector system than the one based on adenovirus was needed for in vivo gene therapy of human diseases that require long-lasting transgene expression. Among the other viruses that were explored as vectors for in vivo gene therapy, one emerged as the most promising for its unique combination of low immunogenicity and high transduction levels in nonreplicating tissues.

The first recombinant adeno-associated viral (AAV) vectors were generated by the group of Muczyczka and Samulski at the University of Florida ⁸¹ based on the rationale that parental AAV vectors are naturally replication-deficient in the absence of helper viruses and that no human diseases were associated with AAV infection. AAV is a small nonenveloped virus that contains a short single-stranded DNA genome of about 5 kb. ⁸² The recombinant vector only contains the inverted terminal repeats and no viral coding sequences. ⁸² The strengths and weaknesses of this Trojan horse reside in its small size.

On one hand, it crosses multiple barriers, including endothelial cells that line vessels ⁸³ and the blood–brain barrier. ⁸⁴ This unique ability has been instrumental to generate effective therapies for untreatable deadly diseases, such as spinal muscular atrophy or Duchenne muscular dystrophy (DMD) where affected tissues are hard to transduce due to the presence of physical barriers and/or require body-wide targeting that can be achieved following a simple intravenous administration. On the other hand, AAV does not package genomes larger than 5 kb efficiently, which prevents its use in gene therapy for conditions that require the delivery of large coding sequences.

Over the last 20 years, a number of improvements to the original AAV serotype 2 (AAV2) delivery system have been brought forward, including among others, (1) the discovery of dozens of natural AAV serotypes and the development of engineered capsid variants through a rational design or directed evolution that provide a unique portfolio of vectors with different tropisms and transduction characteristics, ⁸⁵ (2) self-complementary AAV vectors that contain a double-stranded, shorter genome, which overcomes the second-strand synthesis limitation step for transduction, ⁸⁶ and (3) dual AAV vectors that deliver larger genes split into two halves each independently packaged in AAV that are then reconstituted in a single larger gene ⁸⁷ or larger protein ⁸⁷ in target cells upon coinfection by both dual vectors, which doubles AAV transfer capacity.

The first in vivo gene therapy product approved in the Western world was Glybera in 2012, an AAV1 delivered intramuscularly in patients with a monogenic form of hyperlipidemia ⁸⁸ (Table 1). The combination of the high cost of treatment and the rarity of the condition led to Glybera market withdrawal after only one patient received the approved drug. ⁸⁹ This was the first alarm pointing to the complex challenge of the sustainability of gene therapies.

It took a few more years to get to the first AAV-based ATMP approved both by EMA and FDA. This was the case of Luxturna, an AAV2 vector delivered intraocularly for the treatment of an inherited severe form of childhood blindness (Table 1). ⁹⁰ This success was built on a multiyear effort from the University of Pennsylvania/Children's Hospital of Philadelphia at the beginning of 2000s with a milestone proof-of-concept in a dog model of Leber congenital amaurosis type 2 (LCA2). ⁹¹ This led to a successful multicenter phase I/II clinical trial ⁹² followed by a phase III trial, which unequivocally showed the efficacy of the treatment in addition to its safety. ⁹²

Another major breakthrough was the accelerated approval of Zolgensma, a life-saving gene therapy based on intravenous delivery of high doses of self-complementary AAV9 for treatment of type 1 spinal muscular atrophy (SMA1; Table 1). In the phase I/II study that led to approval, all SMA1 patients survived beyond 2 years of age at which patients with their genotype typically die, and some of them reached and maintained critical psychomotor milestones, which have never been achieved before, with most improvement observed in patients treated in the first few weeks/months after birth. ⁹³ This highlighted the need for newborn screening to allow early intervention in those lethal and progressive conditions for which a therapy becomes available.

In the last 2–3 years, several AAV-based products have been additionally approved, including one for a monogenic form of Parkinson's disease based on local intracerebral delivery of the therapeutic vector, Upstaza (Table 1), ⁹⁴ and two AAVs for hemophilia A (Roctavian) and hemophilia B (Hemgenix). These are provided as a single intravenous administration of the vector that predominantly targets the liver, which is thus converted in a factory for systemic long-term secretion of the clotting factor, therefore eliminating the need for prophylactic or therapeutic infusions of the protein ^95,96 (see review from T. Vandendriessche in this issue of Human Gene Therapy and Table 1).

Another AAV vector that is given as a single intravenous injection at high doses to target muscle in patients with DMD has been approved in June 2023, Elevidys (Table 1). ⁹⁷ It has taken decades to reach this landmark approval given the challenges posed by the large size of the dystrophin coding sequence, which had to be reduced to fit in a single AAV vector, and the need to target muscles body-wide for the therapy to be effective.

Very recently, a herpes simplex virus type 1 (HSV-1) vector has received approval for commercialization in US for the treatment of both the dominant and recessive forms of dystrophic epidermolysis bullosa (DEB) with mutations in the collagen type VII alpha 1 chain (COL7A1) gene. Vyjuvek is a topically applied HSV-1 vector generated by Krystal Biotech that can transduce keratinocytes and fibroblasts, thus restoring the expression of collagen type VII in patients with DEB ⁹⁸ (Table I).

In addition to viral-based ATMPs that are currently on the market, several non-viral gene therapy ATMPs were approved since 2016 for the treatment of rare genetic diseases (Table 1). Spinraza is an antisense oligonucleotide that is administered intrathecally in patients with spinal muscular atrophy, enabling the SMN2 gene to produce the full-length protein, thus replacing the missing SMN protein. A number of small-interfering RNA products have also received commercial authorization (Table I). These include, Onpattro, an siRNA liposome that down-regulates the expression of defective transthyretin, thereby reducing the formation of amyloids and relieving the symptoms of hereditary transthyretin (hATTR) amyloidosis, and two others Givosiran and Oxlumo for acute hepatic porphyria and primary hyperoxaluria type I, respectively.

As happened in the field of ex vivo gene therapy, new viral and nonviral vectors are now in advanced clinical trials for the in vivo treatment of genetic and acquired diseases. For those that require systemic administrations of high doses of AAV vectors such as SMA1 or DMD, rare acute severe adverse events, including thrombotic microangiopathy (TMA) and deaths have been reported that are due to the immunogenicity of high AAV capsid loads. ⁹⁹ These events were observed more consistently in a clinical gene therapy trial for X-linked myotubular myopathy where an underlying liver disease was present. ¹⁰⁰

In vivo genome editing is currently being explored for therapeutic purposes, and the retina has been at the forefront of this development given its favorable risk/benefit. The first-in-human of CRISPR-Cas9 has occurred in patients with a rare form of childhood blindness, Leber congenital amaurosis type 10. ¹⁰¹ Despite promising clinical safety data, the sponsor has discontinued investments in this and other ocular indications. In the case of liver diseases, the efficient targeting of hepatocytes following a single systemic administration of CRISPR-Cas9-lipid nanoparticles has resulted in the significant phenotype improvement of patients with transthyretin amyloidosis, ¹⁰² suggesting that the time is ripe for further implementation of liver genome editing with nonviral delivery methods.

CONCLUDING REMARKS

Advances in the generation of integration competent vectors have markedly improved the efficacy and also the safety associated with ex vivo HSC gene therapies during the last decade. Consequently, several gene therapy HSC-based ATMPs have obtained marketing authorization in Europe and in the United States (Table 1). Following the first European marketing authorization for Strimvelis (HSCs transduced with ADA-RVs; Orchard therapeutics, EU-2016), additional ATMPs consisting of LV-transduced HSCs have also obtained marketing approval in different countries. This is the case for Zynteglo (HSCs transduced with the LentiGlobin BB305; bluebird bio, EU-2019 and US-2022) for the treatment of β-thalassemia, and also for Libmeldy (HSCs transduced with the ARSA-LV for MLD; Orchard Therapeutics, EU-2020) and for Skysona (HSCs transduced with the ABCD1-LV for CALD; bluebird bio, EU-2021 and US-2022).

Unfortunately, some of these ATMPs (Zynteglo and Skysona) have been recently discontinued in Europe because of commercial disagreements. Also, the SCID-ADA and WAS gene therapy programs will probably be discontinued due to commercial issues. The complexity and thus the cost of these ex vivo gene therapies have raised unexpected alarms in their application to diseases with unmet clinical needs.

In addition to ex vivo gene therapy, in vivo gene therapy has shown to be a safe and an effective therapy in humans. However, also some challenges remain to make this transformative therapeutic approach widely available. One is the high prevalence of preexisting anti-AAV immunity in humans who are natural hosts for this virus. ¹⁰³ This is being approached by a variety of strategies, including pretreatment with IgG-cleaving enzymes that transiently deplete neutralizing antibodies that would otherwise block AAV infection following their systemic administration. ¹⁰⁴ Another is the human-specific cell-mediated immune response to AAV vector-transduced cells, which results in their elimination. This can be resolved in several cases by administration of a short regimen of oral corticosteroids. ¹⁰³

History has taught us that it takes decades to characterize the safety and efficacy of a gene delivery platform such as AAV before it can be used reliably in patients. Although the accelerated path of anti-COVID-19 vaccines has shown that this time can be significantly shortened, only time will tell if there will be a paradigm shift in in vivo gene therapy from AAV to nonviral vectors.

As it is the case with ex vivo gene therapies, the complexity and high costs associated with the large-scale manufacturing of viral vectors used for in vivo gene therapy currently pose challenges to the sustainability of this therapeutic modality. The development of easy-to-manufacture nonviral vectors such as lipid and nonlipid nanoparticles similar to the ones used for the COVID-19 vaccination, should indeed help in the development of more accessible gene therapy ATMPs. Additionally, the combination of these new delivery systems together with the revolution of versatile and precise approaches of genome editing should further enhance the implantation of personalized gene therapy in the clinic.

Common efforts from pharmaceutical companies, scientists, clinicians, and regulators are required to find imaginative approaches that would improve the accessibility of affordable gene therapy medicinal products for the treatment of a much higher proportion of patients with rare diseases. ^105,106