Recombinant human acid beta-glucosidase

Gaucher disease is a rare pathology caused by the lysosomal accumulation of sphingolipids, particularly glucosylceramides. The disorder is caused by recessive mutations of the autosomic gene GBA coding for acid beta-glucosidase; this enzyme is a membrane-associated glycoprotein hydrolysing glucosylceramides into glucose and ceramide, which are then recycled for energetic and structural purposes.

The absence of functional forms of the enzyme GCase determines the accumulation of glucosylceramides, particularly in the endothelial reticulum of macrophages and in the central nervous system (neuropathic forms of the disease). Gaucher disease is currently treated at very high costs by enzyme replacement therapy; of note, the regular intravenous infusion of the enzyme results in non-neuropathic patients in the remission of clinical symptoms and a remarkable improvement of life.

Project advancement

The technological platform is now in the licensing phase to an European pharmaceutical industry. The enzyme is produced in rice endosperm with the GeneHPE technology. All steps concerning the achievement of the molecular vector, plant transformation and selection of the productive strain have been completed. Best conditions for enzyme extraction and purification according to industrially-scalable systems have been determined. Moreover, a Seed Seed Bank has been established. Pre-clinical in vitro studies have ascertained the structural and functional bioequivalence of the enzyme in respect to the reference drug. Preclinical in vivo studies (rat, primate) have demonstrated the absence of any modification of vital parameters recorded after the infusion of enzyme doses 10-fold higher than those used in human therapy. Pharmacokinetic studies carried out with the enzyme and the reference drug have provided similar results. Transactiva is currently able to produce about 1 ton of dehydrated, high-quality rice endosperm per year; this amount is sufficient for the treatment of approximately 300 patients.

Recombinant human acid maltase

Acid maltase (Aka, acid alpha-glucosidase) deficiency causes an autosomal recessive disease known as glycogen storage disease type II or Pompe disease. It reflects a glycogen build-up in lysosomes and cytoplasm with consequent cellular injury. The disease derives from recessive mutations at the GAA locus; more than 195 mutations have been described to date which determine a variable manifestation of the disease, depending upon the presence/absence of some residual enzyme activity. Typical symptoms are cardiomegaly, cardiomyopathy, hypotonia, respiratory distress and muscular weakness. Glycogen storage disease Type II is currently treated at very high costs by enzyme replacement therapy; the regular infusion of the recombinant enzyme prolongs ventilator-free survival and clearly improves the outcomes in the six-minute walk test. Early diagnosis and therapy is highly beneficial.

Project advancement

The production platform can be licensed to pharmaceutical industries. The enzyme is produced in rice endosperm with the GeneHPE technology. Best conditions for extraction and purification of the enzyme according to industrially-scalable systems have been established. The enzyme is supplied as a glycosylated, 100-kDa precursor. In-vitro analyses have shown that the enzyme is active and harbors the expected N-terminus. Preclinical assays on human GAA-/- fibroblasts have demonstrated that the enzyme is uptaken by cells similarly to the reference drug. Preclinical studies carried out in vivo on knockout 6neo/6neo mice have also demonstrated that both the enzyme and the reference drug are captured by target tissues; however, the enzyme has shown higher levels of uptake in myocardium and diaphragm.

Recombinant human alpha-galactosidase A

Vascular endothelial cells are very sensitive to the build-up of globotriaosylceramide (Gb3). Gb3 accumulation within the blood vessels leads to an impairment of their functions. Common complications are high blood pressure, cardiomyopathy and an increased risk of heart disease. Another organ typically affected by Gb3 accumulation is kidney which progressively loses its functionality; renal insufficiency and renal failure are a common cause of death due to the disease. Other symptoms caused by this sphingolipidosis, also known as Fabry disease, are deeply worsening life quality of the patients. Fabry disease is currently treated at very high costs by enzyme replacement therapy.

Project advancement

Transactiva is developing the platform for enzyme production in rice endosperm making use of a synthetic promoter. Through the combination of suitable control elements of gene expression, the content of alpha-galactosidase A has reached levels of absolute convenience. The selection of the productive strain is presently ongoing. Preclinical studies have already demonstrated the functional and structural bioequivalence of the enzyme in respect to the reference drug. The technology is available for licensing to pharmaceutical industries.

Idiotype-specific vaccines against non-Hodgkin lymphomas

One of the most exciting applications of plant biotechnology is the industrial production of antitumoral, patient-specific vaccines. Transactiva has already defined a technological platform in this field for the rapid production of anti-idiotype vaccines for the therapy of peripheral centrofollicular non-Hodgkin lymphomas.

Non-Hodgkin lymphomas are tumors of the lymphatic system which arise from the malignant transformation of B- or T-lymphocytes and are often incurable. Peripheral centrofollicular non-Hodgkin lymphomas are one of the most frequent histotypes in the European Union (about 25% of total lymphomas). In the last decades, the annual incidence of these lymphomas has dramatically increased from 6 to 14 new cases/100.000 inhabitants.

Protocols currently used against non-Hodgkin lymphomas involve chemotherapy and radiotherapy; these methods are only partially effective and cause severe adverse effects. Secondly, a frequent limit of these treatments is that relapsed lymphomas appear and patients need to be retreated.

Transactiva is willing to fill the demand of additional drugs for relapse reduction or delaying through the production of patient-specific vaccines, as well as the demand of more selective (patient-tailored), effective and safe drugs.

Project advancement

Transactiva is exploiting the VIS technology for the production of patient-specific vaccines, which is based on minibody engineering in tobacco. This technology has been licensed to an European pharmaceutical industry. The VIS minibody combines the three typical components of an antitumoral vaccine in a single polypeptide chain: the tumor marker (idiotype), the carrier and the adjuvant. Transactiva has developed an expression vector specifically adapted to the synthesis of biopharmaceuticals in tobacco leaf tissues. High levels of transgene expression are warranted by the use of a novel method of gene design and of a patented 5’-UTR sequence. The company has devised a fast, reliable and highly-performing transformation technique (less than 65 days) in combination with an innovative hydroponic system. The overall facility meet the requirements for full traceability, confinement and management of the biomass and can be easily expanded according to different production scales. Transactiva is currently engaged in the constitution of a hybrid variety for the exploitation of heterosis; the final objective is to prepare a patient-specific vaccine starting from the biomass of a single plant. Preclinical studies carried out in Balb/c with the syngenic BCL-1 lymphoma model have shown that the minibody triggers a strong immunogenic response against the idiotype. Furthermore, pharmacological studies in Balb/c with tumor challenge are under completion.

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