Human Interferon Alpha-2b Gene Research Articles

The Evolution of Nadofaragene Firadenovec: A Review and the Path Forward

BACKGROUND: The intravesical gene therapy nadofaragene firadenovec (rAd-IFNα/Syn3) was FDA approved in 2022 for non-muscle invasive bladder cancer (NMIBC) unresponsive to frontline treatment with BCG, and the first gene therapy developed for bladder cancer. This non-replicating recombinant adenovirus vector delivers a copy of the human interferon alpha-2b gene into urothelial and tumor cells, causing them to express this pleotropic cytokine with potent antitumor effects. OBJECTIVE: To provide a historical overview describing how several decades of preclinical and clinical studies investigating the role of interferon in the treatment of bladder cancer ultimately led to the development of gene therapy with nadofaragene for NMIBC. METHODS: We conducted a review of the literature using PubMed, Google Scholar, and ClinicalTrials.gov to summarize our knowledge of the evolution of interferon-based therapy in NMIBC. RESULTS: The FDA approval of this therapy represents an important landmark in urologic oncology and several decades of research dedicated to the study of interferon’s direct and indirect antitumor properties in NMIBC. The data gathered from the phase 1, 2, and 3 clinical trials continue to provide additional insights into the precise mechanisms underlying both the efficacy of and resistance to nadofaragene. CONCLUSIONS: Nadofaragene leverages the cytotoxic, anti-angiogenic, and immune-modulatory roles of interferon to effectively treat NMIBC that is resistant to BCG. Ongoing studies of resistance mechanisms and prognostic biomarkers have been promising; these will ultimately improve patient selection and allow for the modulation of factors in the tumor or immune microenvironment to further increase therapeutic response.

Absence of mutations in the human interferon alpha-2b gene in workers chronically exposed to ionising radiation.

Individuals chronically exposed to low-level ionising radiation (IR) run the risk of harmful and long-term adverse health effects, including gene mutations and cancer development. The search for reliable biomarkers of IR exposure in human population is still of great interest, as they may have a great implementation potential for the surveillance of occupationally exposed individuals. In this context, and considering previous literature, this study aimed to identify mutations in the human interferon alpha-2b (hIFNα-2b) as a potential biomarker of occupational chronic low-dose IR exposure linking low-IR exposure to the effects on haematopoiesis and reduced immunity. The analysis was performed in the genomic DNA of 51 uranium miners and 38 controls from Kazakhstan, and in 21 medical radiology workers and 21 controls from Italy. hIFNα-2b gene mutations were analysed with the real-time polymerase chain reaction (PCR) or Sanger sequencing. However, none of the investigated workers had the hIFNα-2b mutation. This finding highlights the need for further research to identify biomarkers for early detection of health effects associated with chronic low-dose IR exposure.

Response Surface Optimization of the Expression Conditions for Synthetic Human Interferon alpha-2b Gene in Escherichia coli

Recombinant human interferon alpha-2b is an FDA-approved drug for monotherapy or in combination therapy with other drugs for hepatitis and cancers. It belongs to a family of homologous proteins involved in antiviral, antiproliferative, and immunoregulatory processes. A different expression system has been used for overexpression of this protein. Escherichia coli expression system is a highly characterized host and various expression settings have been developed based on its properties. However, finding the best conditions for the overexpression of recombinant human interferon alpha-2b remains to be addressed. In this study, the expression of synthetic human interferon alpha-2b gene in E. coli was greatly improved by adjusting the expression condition. In this regard, a recombinant gene was designed and codon optimized for the periplasmic expression of this protein. Then, gene subcloning was employed to insert the synthesized gene into the pET22b expression vector. Thereafter, the response surface methodology method was employed to design 20 experiments to find out the optimum points for isopropyl β-D-1-thiogalactopyranoside concentration, post-induction period, and the cell density of induction (OD600). The expression fluctuations were assessed by using the real time polymerase chain reaction method. Our results indicated that the synthetic human interferon alpha-2b gene was successfully codon optimized and subcloned into the expression vector. The real time polymerase chain reaction results revealed that the optimum levels of the selected parameters are 0.27 mM for isopropyl β-D-1-thiogalactopyranoside concentration, 7.98 H for the post-induction period, and 3.93 for cell density (OD600). These optimized conditions led to a 3.5-fold increase in the rhIFNα2b expression, which is highly promising for large scale rhIFNα2b overexpression.

Obtaining of the transgenic Heliantus tuberosus L. plants, callus and “hairy” root cultures able to express the recombinant human interferon alpha-2b gene

This work is the first to our knowledge to describe the successful attempt of Agrobacterium rhizogenes-mediated transformation of topinambour in order to obtain the transgenic H. tuberosus plants, callus and "hairy" root cultures. The plasmid vectors contained the sequence of interferon gene fused with Nicotiana plumbagenifolia L. calreticulin apoplast targeting signal driven by 35S CaMV promoter or root-specific Mll promoter. Nearly 75% isolated Ri-root lines and callus cultures were proved (by PCR analysis) to contain HuINFa-2b transgene. We also managed to obtain H. tuberosus transgenic plants through somatic embryogenesis on the transgenic "hairy" root culture. The obtained transgenic H. tuberosus cultures exhibited high-level antiviral activity that ranged from 2000 to 54500 IU/g FW that makes this crop considered a promising source of recombinant interferon alpha 2b protein.

Molecular analysis of human interferon alpha 2b gene in blood cancers

Molecular analysis of human interferon alpha 2b gene in blood cancers

Mutations of the human interferon alpha-2b gene in brain tumor patients exposed to different environmental conditions.

This study was aimed at finding out mutations of an anticancerous, antiviral and immunomodulatory gene (human interferon alpha-2b) in low- and high-grade brain tumor patients and correlate from hematological profiles. A molecular analysis was performed in which DNAs were extracted from brain biopsy samples of brain tumor patients. The gene was amplified through the PCR technique, and genetic data from sequencing were analyzed by bioinformatics to determine how mutations will lead to changes in human interferon alpha 2b protein in patients. A total of 38% gene mutations were identified among brain tumor patients. The highest percentage of (36%) frameshift mutations was identified. Hematological analysis shows modulations in the 'lymphocytes' parameter in a majority (64%) of the brain tumor patients. Environmental factors have been reported as risks of brain cancer. Patients were found to be under environmental stress from contaminated drinking water and from local gamma radiations. Brain tumor patients were found to have various mutations in an immunomodulating human interferon alpha-2b gene. These patients had immunosuppression that was further affirmed from their hematological profiles. This analysis may be helpful to develop certain biomarkers that may be used to develop novel immunotherapeutical drugs, which enhance a better immune response.

Mutations of the human interferon alpha-2b (hIFN-α2b) gene in occupationally protracted low dose radiation exposed personnel

Ionizing radiations impact human tissues by affecting the DNA bases which constitute genes. Human interferon alpha 2b gene synthesizes a protein which is an important anticancerous, immunomodulatory, anti-proliferative and antiviral protein. This study was aimed to identify interferon alpha-2b mutations as a consequence of the use of occupational chronic low dose radiation by hospital radiation exposed workers. A molecular analysis was done in which DNAs were extracted from blood samples from radiology, radiotherapy and nuclear medicine workers. The gene was amplified through polymerase chain reaction and further genetic data from sequencing results analyzed by bioinformatics tools in order to determine as to how mutations in interferon alpha 2b sequences will lead to changes in human interferon alpha-2b protein. A total of 41% gene mutations was detected among all radiation exposed workers in which higher percentage (5.4%) of base insertion mutations and 14% frameshift mutations were found in radiology workers. The chronic use of low dose of radiations by occupational workers has a significant correlation with mutational effects on interferon alpha 2b gene, further evident by depressed interferon alpha levels in serum. This can lead to depressed immunity in radiation exposed workers. Hematological profiling of this group also showed hyperimmune response in the form of lymphocytosis.

Superoxide dismutase activity in transgenic canola

Superoxide dismutase (SOD) activity was investigated in leaves of transgenic canola plants which expressed heterologous genes of different origin, namely 1) herbicide resistance genes (bar and simultaneously bar and epsps); 2) DesC desaturase gene (desC) of cyanobacterium Synechococcus vulcanus; 3) human interferon alpha2b gene (huLFN-alpha2b); 4) esxA::fbpB(deltaTMD) fused gene, encoding ESAT-6 and Ag85b Mycobacterium tuberculosis proteins, inducing immune response against tuberculosis; 5) cyp11A1 gene of cytochrome P450(scc) from bovine adrenal cortex mitochondria. Introduction of herbicide resistance genes as well as desaturase gene of cyanobacterium and mycobacterium's genes did not change leaf SOD activity. At the same time it was shown that cyp11A1 and huIFN-alpha2b canola have increased leaf SOD activity up 58 and 33%, respectively, compared with control ones in non-stress conditions. It may be a prerequisite for improved resistance of these plants to the stressors of different origin.

Antiviral activity of extracts of transgenic chicory and lettuce plants with the human interferon α2b gene

Biological activity of protein extracts from transgenic plants of chicory Cichorium intybus L. and lettuce Lactuca sativa L. with human interferon alpha2b gene was investigated against vesicular stomatitis virus. It was shown that the extracts from the hairy roots of chicory and lettuce transformed by A. rhizogenes possess the antiviral activity 1620...5400 IU/g weight, and the extracts from leaves of the plants transformed by A. tumefaciens--till 9375 IU/g weight. Dependence of plant extract biological activity on the transformation vector was shown.

Multiplex PCR assay for detection of human interferon alpha2b gene in transgenic plants

During the last decade interferons are regarded as potent candidates for generation of plant-based edible vaccines because of broad spectrum of antiviral activities and adjuvant properties. Establishment and certification of numerous interferon producing plant systems requests development of fast and efficient multiplex PCR protocol for the transgene detection in GM plants. Here we represent a protocol for simultaneous amplification in one assay of fragments of hIFN alpha 2b gene and two control genes, namely virD1 of Agrobacterium tumefaciens and conservative region of plant actin gene.

Toxicity and exposure of an adenovirus containing human interferon alpha-2b following intracystic administration in cynomolgus monkeys

The safety and toxicokinetics of SCH 721015, an adenovirus encoding the human interferon alpha-2b gene, and Syn3 (SCH 209702), a novel excipient, were assessed in cynomolgus monkeys administered intravesical doses of 2.5 × 10E11 or 1.25 × 10E13 particles SCH 721015 in 25 mg Syn3 or 25 mg Syn3 alone on study days 1 and 91. There was no systemic toxicity. Monkeys dosed with SCH 721015 in Syn3 were positive for SCH 721015-specific DNA in the urine for 2 to 3 days following each dose and had interferon alpha-2b protein in the urine for 1-3 days after a single dose and in fewer animals after a second dose. Intracystic administration was associated with inflammation and focal/multifocal ulceration in the urinary bladder and irritation in the ureters and urethra at necropsy. The physical trauma from catheterization and filling/emptying of the bladder was likely a contributing factor and Syn3 exacerbated the trauma. There was nearly complete resolution of these findings 2 months after the last dose. The trauma to the bladder likely contributed to low, transient systemic exposure to Syn3, SCH 721015 and human interferon protein. The results of this study support the clinical investigation of SCH 721015 in Syn3.

High-level expression of human interferon alpha-2b in transgenic carrot (Daucus carota L.) plants

In this study, we report the obtaining of carrot plants expressing human interferon alpha-2b via Agrobacterium-mediated transformation using two vector constructs containing the sequence coding for interferon gene fused with Nicotiana plumbagenifolia calreticulin apoplast targeting signal driven by 35S CaMV promoter and root-specific Mll promoter. The human interferon alpha-2b gene was correctly translated in carrot plants according to Western blot analysis. The recombinant protein exhibited antiviral activity in vitro by inhibition of vesicular stomatitis virus replication in established piglet testicular cells. The results demonstrated the higher activity of interferon accumulated in carrot plants for young leaves (up to 50.7 × 10(3) IU/g FW) compared to the mature ones probably due to the degradation-susceptible nature of this protein. The taproot-expressing system could have also provided the sufficient protein amounts (up to 16.5 × 10(3) IU/g FW) and could possibly be used for generating interferon alpha-2b protein in planta for preventing and curing infectious diseases.

Production of human interferon ALPHA 2b in plants of Nicotiana excelsior by Agrobacterium-mediated transient expression

Human interferon alpha2b gene was transiently expressed in Nicotiana excelsior plants. Fusion with N. plumbaginifolia calreticulin signal peptide for improved apoplast targeting and carrying out the expression under optimized conditions resulted in maximal interferon activity of 3.2 x 10(3) IU/g fresh weight (FW) with an average of 2.1 +/- 0.8 x 10(3) IU/g FW. It proves that N. excelsior is a suitable host for Agrobacterium-mediated transient expression of genes encoding physiologically active human proteins. The transient expression conditions optimized for GFP marker protein were confirmed to be preferable for hIFN alpha2b.

Sustained intravesical interferon protein exposure is achieved using an adenoviral-mediated gene delivery system: A study in rats evaluating dosing regimens

Objectives To evaluate whether a recombinant replication-deficient adenovirus containing the secreted human interferon alpha-2b gene (rAd-IFN) could improve the tissue and urine levels of IFN protein by transducing the urothelium with the secreted human IFN-alpha gene. We also assessed whether varying the interval between rAd-IFN/Syn3 treatments would improve the duration and levels of gene expression. Methods The rats received intravesical administration of rAd-IFN at varying concentrations in a formulation containing Syn3, an agent identified that facilitates passage of the adenovirus through the protective barrier of the bladder. Urine was collected daily for 7 days, and human IFN was measured in the urine by enzyme-linked immunosorbent assay. For the redosing studies, the animals received a second dose at varying intervals ranging from 1 to 7 days after the first dose or at longer intervals (30, 60, or 90 days). Results Rats that received intravesical administration of rAd-IFN in a Syn3 formulation expressed levels of human IFN protein in their urine at peak concentrations of 50,000 to 100,000 pg/mL, but were undetectable by 7 days. Expression was localized to the bladder with only minimal systemic exposure to IFN. Short-term redosing marginally improved the IFN urine concentrations, with maximal levels achieved when a second dose was administered 3 days after a first dose. Although gene expression was attenuated when a second dose was given 5 to 7 days after the first treatment, the levels and duration of IFN expression recovered when the interval was increased to 90 days. Conclusions Intravesical treatment with rAd-IFN facilitates high levels of IFN transgene exposure and may be a new approach to treating superficial bladder cancer.