Beta-thalassaemia: from the first trial to a new gene therapy protocol

A new clinical trial is evaluating an optimised ex vivo gene therapy protocol for transfusion-dependent beta-thalassaemia developed at SR-Tiget.

More than ten years after the first pilot study, ex vivo gene therapy for transfusion-dependent beta-thalassaemia is being evaluated in a new clinical trial protocol sponsored by Fondazione Telethon and Ospedale San Raffaele. The trial is designed to assess, in nine patients aged between 3 and 35, the safety and efficacy profile of an optimised version of the gene therapy, developed at SR-Tiget, based on the genetic modification of haematopoietic stem cells using a lentiviral vector, with an initial phase in adults followed by subsequent extension to children and adolescents.

This trial is the result of a complex scientific pathway, starting from the analysis of data generated in the previous 2015 trial, published in Nature Medicine in 2019, and from the dialogue between basic research and applied research.

The new protocol was designed with the aim of improving transduction efficiency while preserving, as far as possible, the stem-cell properties of the cells during ex vivo manipulation. The introduction of a functional gene using a lentiviral vector remains the foundation of the approach; optimisation concerns the way in which the cells are prepared before infusion.

Beta-thalassaemia, together with sickle cell disease, was one of the first diseases for which a correlation between a genetic defect and the disease itself was established. However, the clinical application of genetic correction strategies came later than for other, much less common conditions. The reason lies in the biological complexity of transfusion-dependent beta-thalassaemia and in the regulation of gene expression including the need to achieve adequate and sustained production of functional haemoglobin.

In this condition, simply introducing a working copy of the beta-globin gene into haematopoietic stem cells may not be sufficient to achieve a meaningful clinical effect. The potential therapeutic effect depends on multiple parameters, including the ability of reaching a high amount of protein produced, and therefore on transduction efficiency, the level of transgene expression and the proportion of corrected cells that contribute stably to haematopoiesis.  

In gene therapy, the aim is to come close to physiological production capacity by introducing a sufficient number of vector copies into an adequate proportion of haematopoietic stem cells. “Having two copies in 10% of cells would not correct anything” observes Giuliana Ferrari, Head of the Gene Transfer into Stem Cells Unit at SR-Tiget and Professor of Molecular Biology at Vita-Salute San Raffaele University. “The ideal would be to have two copies in 100% of cells”.

The first trial, carried out in 2015 at Ospedale San Raffaele in Milan, helped clarify precisely this point: which characteristics the cell product needed to have for genetic correction to translate into a clinical benefit. “We learnt what is needed to make a patient transfusion-independent and what, instead, did not work when patients did not achieve a complete clinical benefit, in relation to the product we had developed: cells genetically modified with GLOBE, the vector produced and designed in our laboratories” explains Ferrari.

The new protocol stems from this work: from the effort to optimise these product parameters,  with the clinical objective of evaluating wherever such changes may increase the likelihood of achieving clinically relevant outcomes, including transfusion independence.

To support the optimisation of the product, researchers at SR-Tiget modified several steps in the cell culture and preparation process. To improve gene transfer, they tested a number of molecules known as transduction enhancers: “These are biologically known compounds, but they had never been tested before for this use. They must not alter the biological nature of the cell, but at the same time they need to allow greater interaction with the vector and greater integration of the genetic material being delivered” continues Ferrari.

A second intervention concerned the culture time. “The cells collected from patients, in order to be genetically modified, have to be cultured under specific conditions. In the previous protocol, the culture time was more than 72 hours; today, with the new product, we have shortened the time of exposure to the vector by at least one day” explains Ferrari. This step is particularly important when working with haematopoietic stem cells. These cells must be kept as undifferentiated as possible ex vivo in order to preserve their ability to engraft and reconstitute haematopoiesis once reinfused.

These modifications are intended to generate a gene therapy product with potentially improved characteristics and they concern the manufacturing steps that take place before infusion. Once reinfused in the patient, the cells must engraft in the bone marrow, reconstitute haematopoiesis and contribute to erythropoiesis. Even when the product contains a high percentage of transduced cells and an adequate number of vector copies, the proportion of stem cells that will actually engraft and persist over time may vary depending on the characteristics of individual patients. The transition from the product prepared in the laboratory to the behaviour of the cells in the patient remains one of the key variables to be assessed in the clinical setting.

When genetically corrected cells are reinfused into the patient, they must reach the bone marrow and engraft in the bone marrow niche, the microenvironment that supports haematopoietic stem cells and contributes to the reconstitution of haematopoiesis. In beta-thalassaemia, however, this environment may be altered by the history of the disease itself.

Indeed, the complexity of the disease does not depend solely on the genetic mutation that compromises beta-globin production. Chronic anaemia keeps the bone marrow in a state of continuous stimulation to produce red blood cells, but erythropoiesis remains ineffective. Added to this is iron overload, linked both to the disease and to the patient’s transfusion history, which can exert a toxic effect on the cellular components of the bone marrow.

The result is a chronically stressed environment. “The bone marrow niche is not a hospitable environment: it is an environment stressed by several biological signals and by iron accumulation” observes Ferrari. This state of stress can contribute to making the conditions required for engraftment less favourable.

Ferrari’s laboratory has devoted a significant part of its basic research to this topic, playing a pioneering role. For a long time, in beta-thalassaemia and sickle cell disease, the role of the bone marrow niche received limited attention, from both a clinical and a biological perspective. Investigating this dimension means shifting the focus from the corrected cell alone to the biological context in which that cell must function. The new protocol acts on the cell product, improving the conditions under which the cells are corrected and prepared; the niche, by contrast, remains a biological component of the patient, one that needs to be better understood because it may contribute to the quality and duration of the response.

It is precisely on this front that a research perspective is being explored. In the murine model, Ferrari’s group observed the alteration of a factor normally produced by the body and linked to the functioning of the niche. This led to the experimental hypothesis of a combined therapeutic approach, in which gene therapy could potentially be associated with a protein factor intended to make the bone marrow environment more favourable to the engraftment of corrected cells. “We are working to understand whether it may be possible to propose a combined therapy: gene therapy associated with this factor, which could help create a better niche” explains Ferrari.

The value of this line of research lies above all in broadening our understanding of the biological factors that can influence the success of advanced therapies: not only the quality of the corrected cells, but also the conditions of the environment in which those cells must engraft and function over time.

The therapeutics landscape has evolved substantially since the first beta-thalassaemia study was launched. In recent years, advanced therapies have progressed significantly, and gene editing has already shown clinically relevant results in transfusion-dependent beta-thalassaemia. Within this landscape, the investigational approach developed at SR-Tiget represents a distinct strategy, based on a distinct molecular rationale and long-standing experience with lentiviral vectors.

What distinguishes these strategies first and foremost is their molecular mechanism. Ex vivo gene therapy using a lentiviral vector is a gene addition strategy. Gene editing, by contrast, follows a different logic: it does not add a copy of the beta-globin gene, but acts on the mechanisms that regulate foetal haemoglobin expression, reactivating its production. Precisely because the mechanisms are different, any comparison between the two strategies must remain cautious. Therapy using lentiviral vectors involves the stable integration of the vector into the genome and therefore requires monitoring of the integration sites, in order to control the potential risk of insertional mutagenesis. Gene editing, instead, is based on a targeted cut in the genome; in this case too, there are theoretical risks, linked to possible unintended cuts, which must be anticipated, tested and monitored over time.

“These are two different molecular mechanisms, so any comparison is scientifically difficult. Each therapy has its own benefit-risk profile; what matters is that the risks are predicted, known and monitorable” Ferrari stresses.

Another difference concerns the history of the technologies themselves: lentiviral vectors have a longer clinical follow-up available across different diseases; gene editing, although it has already reported important clinical results, is a more recent technology and continues to require long-term monitoring.

The point is to recognise that transfusion-dependent beta-thalassaemia remains a disease with a broad and heterogeneous therapeutic need. The number and diversity of patients who may potentially be eligible for definitive therapies, particularly in areas of the world where prevalence is higher, suggests that different strategies may need to be evaluated to address different clinical contexts and patient profiles.

It is in this context that the new protocol developed at SR-Tiget is entering clinical testing. “The laboratory studies and preclinical analyses produced very encouraging results, enough to convince us to propose a different product, ten years after the treatment of the first patient” explains Ferrari. After years of work on the cell product, the process was scaled up in SR-Tiget laboratories and then transferred to the pharmaceutical facility of Ospedale Pediatrico Bambino Gesù dedicated to advanced therapies, where production will take place. The new phase IIb trial will assess whether the modifications introduced in the laboratory — including improved transduction efficiency, reduced culture time and preservation of stemness — can translate, in patients, into clinically meaningful benefit, including the possibility of transfusion independence.