Intraparenchymal delivery of adeno-associated virus vectors for the gene therapy of neurological diseases.

INTRODUCTION: In gene therapy with adeno-associated virus (AAV) vectors for diseases of the central nervous system, the vectors can be administered into blood vessels, cerebrospinal fluid space, or the brain parenchyma. When gene transfer to a large area of the brain is required, the first two methods are used, but for diseases in which local gene transfer is expected to be effective, vectors are administered directly into the brain parenchyma. AREAS COVERED: Strategies for intraparenchymal vector delivery in gene therapy for Parkinson's disease, aromatic l-amino acid decarboxylase (AADC) deficiency, and epilepsy are reviewed. EXPERT OPINION: Stereotactic intraparenchymal injection of AAV vectors allows precise gene delivery to the target site. Although more surgically invasive than intravascular or intrathecal administration, intraparenchymal vector delivery has the advantage of a lower vector dose, and preexisting neutralizing antibodies have little effect on the transduction efficacy. This approach improves motor function in AADC deficiency and led to regulatory approval of an AAV vector for the disease in the EU. Although further validation through clinical studies is needed, direct infusion of viral vectors into the brain parenchyma is expected to be a novel treatment for Parkinson's disease and drug-resistant epilepsy.

Aromatic L-Amino Acid Decarboxylase Deficiency: A Genetic Screening in Sicilian Patients with Neurological Disorders.

Aromatic L-amino acid decarboxylase deficiency (AADCd) is a rare autosomal recessive neurometabolic disorder caused by AADC deficiency, an enzyme encoded by the DDC gene. Since the enzyme is involved in the biosynthesis of serotonin and dopamine, its deficiency determines the lack of these neurotransmitters, but also of norepinephrine and epinephrine. Onset is early and the key signs are hypotonia, movement disorders (oculogyric crises, dystonia and hypokinesia), developmental delay and autonomic dysfunction. Taiwan is the site of a potential founder variant (IVS6+4A>T) with a predicted incidence of 1/32,000 births, while only 261 patients with this deficit have been described worldwide. Actually, the number of affected persons could be greater, given that the spectrum of clinical manifestations is broad and still little known. In our study we selected 350 unrelated patients presenting with different neurological disorders including heterogeneous neuromuscular disorders, cognitive deficit, behavioral disorders and autism spectrum disorder, for which the underlying etiology had not yet been identified. Molecular investigation of the DDC gene was carried out with the aim of identifying affected patients and/or carriers. Our study shows a high frequency of carriers (2.57%) in Sicilian subjects with neurological deficits, with a higher concentration in northern and eastern Sicily. Assuming these data as representative of the general Sicilian population, the risk may be comparable to some rare diseases included in the newborn screening programs such as spinal muscular atrophy, cystic fibrosis and phenylketonuria.

Activate the endogenous Cu2+ switch for Zn(DDC)2 liposomes conversion: Providing a safer and less toxic alternative in cancer therapy.

The ancient anti-alcohol drug disulfiram (DSF) has gained widespread attention for its highly effective anti-tumor effects in cancer treatment. Our previous studies have developed liposome of Cu (DDC)2 to overcome the limitations, like the poor water solubility. However, Cu (DDC)2 liposomes still have shown difficulties in severe hemolytic reactions at high doses and systemic toxicity, which have limited their clinical use. Therefore, this study aims to exploratively investigate the feasibility of using DSF or DDC in combination also can chelate Zn2+ to form zinc diethyldithiocarbamate (Zn (DDC)2). Furthermore, this study prepared stable and homogeneous Zn (DDC)2 liposomes, which were able to be released in the tumor microenvironment (TME). The released Zn (DDC)2 was converted to Cu (DDC)2 with the help of endogenous Cu2+-switch enriched in the TME, which has a higher stability constant compared with Zn (DDC)2. In other words, the Cu2+-switch is activated at the tumor site, completing the conversion of the less cytotoxic Zn (DDC)2 to the more cytotoxic Cu (DDC)2 for effective tumor therapy so that the Zn (DDC)2 liposomes in vivo achieved the comparable therapeutic efficacy and provided a safer alternative to Cu (DDC)2 liposomes in cancer therapy.

RNA modification regulator DDC in endometrial cancer affects the tumor microenvironment and patient prognosis.

Uterine corpus endometrial carcinoma (UCEC) is infiltrated by immune cells, which are involved in the growth and proliferation of malignant tumors and resistance to immunotherapy. This study suggested that RNA modification regulators played an important role in the development and prognosis of UCEC. Many studies confirmed that RNA modification played an essential role in tumor immune regulation, and abnormal RNA modification contributed to tumorigenesis and cancer progression. Based on the RNA modification regulatory factors, the UCEC samples from TCGA (The Cancer Genome Atlas) were classified into two clusters, namely Cluster A and Cluster B, using unsupervised consensus clustering. We obtained DEG (differentially expressed genes) between the two clusters, and constructed a risk model of RNA modification-related genes using DEGs. Cluster A had lower RNA modification regulatory factors, richer immune cell infiltration, and better prognosis. The differentially expressed genes between the two clusters were obtained, and these genes were used for modeling. This model divided patients with UCEC into two groups. The low-risk group had better immune infiltration, and the ROC (receiver operating characteristic) curve showed that this model had good predictive efficacy. The low-risk group had a better response to immunotherapy by immune checkpoint prediction. We obtained the key gene L-dopa decarboxylase (DDC) through the intersection of LASSO model genes and GEO dataset GSE17025. We evaluated the potential biological functions of DDC. The differences in the expression of DDC were verified by immunohistochemistry. We evaluated the relationship between DDC and immune cell infiltration and verified this difference using immunofluorescence. Cluster A with low expression of RNA modification regulators has better prognosis and richer immune cell infiltration, therefore, we believed that RNA modification regulators in UCEC were closely related to the tumor microenvironment. Also, the risk score could well predict the prognosis of patients and guide immunotherapy, which might benefit patients with UCEC.

Exome sequencing data screening to identify undiagnosed Aromatic l-amino acid decarboxylase deficiency in neurodevelopmental disorders.

Aromatic l-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive neurometabolic disorder caused by biallelic pathogenic variants in the DDC gene and mainly characterized by developmental delay, hypotonia, and oculogyric crises. Early diagnosis is crucial for correct patient management; however, many patients remain misdiagnosed or undiagnosed due to the rarity and clinical heterogeneity of the disorder especially in the milder forms. Here, we applied exome sequencing approach by screening 2000 paediatric patients with neurodevelopmental disorders to identify possible new AADC variants and AADC deficiency patients. We identified five distinct DDC variants in two unrelated individuals. Patient #1 harboured two compound heterozygous DDC variants: c.436-12T > C and c.435 + 24A>C and presented with psychomotor delay, tonic spasms, and hyperreactivity. Patient #2 had three homozygous AADC variants: c.1385G > A; p.Arg462Gln, c.234C > T; p.Ala78 = , and c.201 + 37A > G and presented with developmental delay and myoclonic seizures. The variants were classified as benign class I variants and therefore non-causative according to the ACMG/AMP guidelines. Since the AADC protein is a structural and functional obligate homodimer, we evaluated the possible AADC polypeptide chain combinations in the two patients and determined the effects resulting from the amino acid substitution Arg462Gln. Our patients carrying DDC variants presented clinical manifestations not precisely overlapped to the classical symptoms exhibited by the most severe AADC deficiency cases. However, screening data derived from exome sequencing in patients featuring wide-range symptoms related to neurodevelopmental disorders may help to identify AADC deficiency patients, especially when applied to larger cohorts.

Gene Therapy for Dopamine Dyshomeostasis: From Parkinson's to Primary Neurotransmitter Diseases.

Neurological disorders encompass a broad range of neurodegenerative and neurodevelopmental diseases that are complex and almost universally without disease modifying treatments. There is, therefore, significant unmet clinical need to develop novel therapeutic strategies for these patients. Viral gene therapies are a promising approach, where gene delivery is achieved through viral vectors such as adeno-associated virus and lentivirus. The clinical efficacy of such gene therapies has already been observed in two neurological disorders of pediatric onset; for spinal muscular atrophy and aromatic L-amino acid decarboxylase (AADC) deficiency, gene therapy has significantly modified the natural history of disease in these life-limiting neurological disorders. Here, we review recent advances in gene therapy, focused on the targeted delivery of dopaminergic genes for Parkinson's disease and the primary neurotransmitter disorders, AADC deficiency and dopamine transporter deficiency syndrome (DTDS). Although recent European Medicines Agency and Medicines and Healthcare products Regulatory Agency approval of Upstaza (eladocagene exuparvovec) signifies an important landmark, numerous challenges remain. Future research will need to focus on defining the optimal therapeutic window for clinical intervention, better understanding of the duration of therapeutic efficacy, and improved brain targeting. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

The treatment of hepatocellular carcinoma with SP94 modified asymmetrical bilayer lipid-encapsulated Cu(DDC)2 nanoparticles facilitating Cu accumulation in the tumor.

BACKGROUND: Copper diethyldithiocarbamate (Cu(DDC)2) has been demonstrated to possess excellent antitumor activity. However, the extremely poor water solubility of Cu(DDC)2 bring difficulty for its formulation research. In this study, we aim to develop a novel nanocarrier for Cu(DDC)2 delivery to overcome this obstacle and enhance antitumor activity. METHODS: The SP94 modified asymmetrical bilayer lipid-encapsulated Cu(DDC)2 nanoparticles (DCDP) was established by combining the method of inverse microemulsion aggregation and thin-film dispersion. In vitro cellular assays and in vivo tumor-xenograft experiments were conducted to evaluate the tumor chemotherapeutic effect of DCDP. And the vital role of copper ions played in DSF or DDC (DSF/DDC)-based cancer chemotherapy was also explored. RESULTS: DCDP with an encapsulation efficiency (EE%) of 74.0% were successfully prepared. SP94 modification facilitated cellular intake for DCDP, and promoted apoptosis to repress tumor cell proliferation (IC50, 200 nM). And DCDP effectively inhibited tumor growth with a high tumor inhibition rate of 74.84%. Furthermore, Cu(DDC)2 was found to facilitate the copper ion accumulation in tumor tissues, which is beneficial to therapy with high potency. CONCLUSION: DCDP exhibited high-efficient tumor chemotherapeutic efficacy and provided a novel strategy for investigating the anticancer mechanism of Cu(DDC)2.

Lack of EGFR catalytic activity in hepatocytes improves liver regeneration following DDC-induced cholestatic injury by promoting a pro-restorative inflammatory response.

Despite the well-known hepatoprotective role of the epidermal growth factor receptor (EGFR) pathway upon acute damage, its specific actions during chronic liver disease, particularly cholestatic injury, remain ambiguous and unresolved. Here, we analyzed the consequences of inactivating EGFR signaling in the liver on the regenerative response following cholestatic injury. For that, transgenic mice overexpressing a dominant negative mutant human EGFR lacking tyrosine kinase activity (ΔEGFR) in albumin-positive cells were submitted to liver damage induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), an experimental model resembling human primary sclerosing cholangitis. Our results show an early activation of EGFR after 1-2 days of a DDC-supplemented diet, followed by a signaling switch-off. Furthermore, ΔEGFR mice showed less liver damage and a more efficient regeneration following DDC injury. Analysis of the mechanisms driving this effect revealed an enhanced activation of mitogenic/survival signals, AKT and ERK1/2-MAPKs, and changes in cell turnover consistent with a quicker resolution of damage in response to DDC. These changes were concomitant with profound differences in the profile of intrahepatic immune cells, consisting of a shift in the M1/M2 balance towards M2 polarity, and the Cd4/Cd8 ratio in favor of Cd4 lymphocytes, overall supporting an immune cell switch into a pro-restorative phenotype. Interestingly, ΔEGFR livers also displayed an amplified ductular reaction, with increased expression of EPCAM and an increased number of CK19-positive ductular structures in portal areas, demonstrating an overexpansion of ductular progenitor cells. In summary, our work supports the notion that hepatocyte-specific EGFR activity acts as a key player in the crosstalk between parenchymal and non-parenchymal hepatic cells, promoting the pro-inflammatory response activated during cholestatic injury and therefore contributing to the pathogenesis of cholestatic liver disease. © 2022 The Pathological Society of Great Britain and Ireland.

Eladocagene Exuparvovec: First Approval.

Eladocagene exuparvovec (Upstaza™) is a gene therapy developed by PTC Therapeutics for the treatment of human aromatic L-amino acid decarboxylase (AADC) deficiency. Eladocagene exuparvovec comprises an adeno-associated virus vector that delivers the dopa decarboxylase (DDC) gene, the gene for human AADC. Eladocagene exuparvovec was approved in July 2022 in the EU for the treatment of patients aged 18 months and older with a clinical, molecular, and genetically confirmed diagnosis of AADC deficiency with a severe phenotype (i.e. patients who cannot sit, stand or walk). This article summarizes the milestones in the development of eladocagene exuparvovec leading to this first approval for the treatment of patients aged 18 months and older with AADC deficiency.

Herbaceous peony AP2/ERF transcription factor binds the promoter of the tryptophan decarboxylase gene to enhance high-temperature stress tolerance.

Global warming has multifarious adverse effects on plant growth and productivity. Nonetheless, the effects of endogenous phytomelatonin on the high-temperature resistance of plants and the underlying genetic mechanisms remain unclear. Here, herbaceous peony (Paeonia lactiflora Pall.) tryptophan decarboxylase (TDC) gene involved in phytomelatonin biosynthesis was shown to respond to high-temperature stress at the transcriptional level, and its transcript level was positively correlated with phytomelatonin production. Moreover, overexpression of PlTDC enhanced phytomelatonin production and high-temperature stress tolerance in transgenic tobacco, while silencing PlTDC expression decreased these parameters in P. lactiflora. In addition, a 2402 bp promoter fragment of PlTDC was isolated, and DNA pull-down assay revealed that one APETALA2/ethylene-responsive element-binding factor (AP2/ERF) transcription factor, PlTOE3, could specifically activate the PlTDC promoter, which was further verified by yeast one-hybrid assay and luciferase reporter assay. PlTOE3 was a nucleus-localized protein, and its transcript level responded to high-temperature stress. Additionally, transgenic tobacco overexpressing PlTOE3 showed enhanced phytomelatonin production and high-temperature stress tolerance, while silencing PlTDC expression obtained the opposite results. These results illustrated that PlTOE3 bound the PlTDC promoter to enhance high-temperature stress tolerance by increasing phytomelatonin production in P. lactiflora.

Sphingomyelin-based PEGylation Cu (DDC)2 liposomes prepared via the dual function of Cu2+ for cancer therapy: Facilitating DDC loading and exerting synergistic antitumor effects.

The old alcohol-aversion drug disulfiram (DSF) has aroused wide attention as a drug repurposing strategy in terms of cancer therapy because of the high antitumor efficacy in combination with copper ion. However, numerous defects of DSF (e.g., the short half-life and acid instability) have limited the application in cancer treatment. Cu (DDC)2, the complex of diethyldithiocarbamate (DDC, DSF metabolite) and Cu2+, have been proven as the vital active component on cancer, which have aroused the attention of researchers from DSF to Cu (DDC)2. However, the poor water solubility of Cu (DDC)2 increase more difficulties to the treatment and in-depth investigations of Cu (DDC)2. In this study, sphingomyelin (SM)-based PEGylated liposomes (SM/Chol/DSPE-mPEG2000 (55:40:5, mole%)) were produced as the carriers for Cu (DDC)2 delivery to enhance the water solubility. DDC was added to Cu-containing liposomes with a higher encapsulation efficiency of more than 90%, and it reacted with Cu2+ to synthesize Cu (DDC)2. Due to the high phase transition temperature of SM and strong intermolecular hydrogen bonds with cholesterol, SM-based liposomes would be conducive to enhancing the stability of Cu (DDC)2 and preventing drug leakage during delivery. As proven by pharmacokinetic studies, loading Cu (DDC)2 into liposomes improve bioavailability, and the area under the curve (AUC0-t) and the mean elimination half-life (t1/2) increased 1.9-time and 1.3-time to those of free Cu (DDC)2, respectively. Furthermore, the anticancer effect of Cu (DDC)2 was enhanced by the liposomal encapsulation, thus resulting in remarkable cell apoptosis in vitro and a tumor-inhibiting rate of 77.88% in vivo. Thus, it was concluded that Cu (DDC)2 liposomes could be promising in cancer treatment.

Melatonin enhances stem strength by increasing lignin content and secondary cell wall thickness in herbaceous peony.

Cut flower quality is severely restrained by stem bending due to low stem strength. Melatonin has been shown to function in many aspects of plant growth and development, yet whether it can enhance stem strength, and the corresponding underlying mechanisms remain unclear. We investigated the role of melatonin in enhancement of stem strength in herbaceous peony (Paeonia lactiflora Pall.) by applying exogenous melatonin and changing endogenous melatonin biosynthesis. Endogenous melatonin content positively correlated with lignin content and stem strength in various P. lactiflora cultivars. Supplementation with exogenous melatonin significantly enhanced stem strength by increasing lignin content and the S/G lignin compositional ratio, up-regulating lignin biosynthetic gene expression. Moreover, overexpression of TRYPTOPHAN DECARBOXYLASE GENE (TDC) responsible for the first committed step of melatonin biosynthesis in tobacco, significantly increased endogenous melatonin, which further increased the S/G ratio and stem strength. In contrast, silencing PlTDC in P. lactiflora decreased endogenous melatonin, the S/G ratio and stem strength. Finally, manipulating the expression of CAFFEIC ACID O-METHYLTRANSFERASE GENE (COMT1), which is involved in both melatonin and lignin biosynthesis, showed even greater effects on melatonin, the S/G ratio and stem strength. Our results suggest that melatonin has a positive regulatory effect on P. lactiflora stem strength.

Characterizing serotonin biosynthesis in Setaria viridis leaves and its effect on aphids.

KEY MESSAGE: A combined transcriptomic and metabolic analysis of Setaria viridis leaves responding to aphid infestation was used to identify genes related to serotonin biosynthesis. Setaria viridis (green foxtail), a short life-cycle C4 plant in the Poaceae family, is the wild ancestor of Setaria italica (foxtail millet), a resilient crop that provides good yields in dry and marginal land. Although S. viridis has been studied extensively in the last decade, the molecular mechanisms of insect resistance in this species remain under-investigated. To address this issue, we performed a metabolic analysis of S. viridis and discovered that these plants accumulate the tryptophan-derived compounds tryptamine and serotonin. To elucidate the defensive functions of serotonin, Rhophalosiphum padi (bird cherry-oat aphids) were exposed to this compound, either by exogenous application to the plant medium or with artificial diet bioassays. In both cases, exposure to serotonin increased aphid mortality. To identify genes that are involved in serotonin biosynthesis, we conducted a transcriptome analysis and identified several predicted S. viridis tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H) genes. Two candidate genes were ectopically expressed in Nicotiana tabacum, where SvTDC1 (Sevir.6G066200) had tryptophan decarboxylase activity, and SvT5H1 (Sevir.8G219600) had tryptamine hydroxylase activity. Moreover, the function of the SvTDC1 gene was validated using virus-induced gene silencing in S. italica, which caused a reduction in serotonin levels. This study provides the first evidence of serotonin biosynthesis in Setaria leaves. The biosynthesis of serotonin may play an important role in defense responses and could prove to be useful for developing more pest-tolerant Setaria italica cultivars.

Long-term efficacy and safety of eladocagene exuparvovec in patients with AADC deficiency.

Aromatic L-amino acid decarboxylase deficiency results in decreased neurotransmitter levels and severe motor dysfunction. Twenty-six patients without head control received bilateral intraputaminal infusions of a recombinant adeno-associated virus type 2 vector containing the human aromatic L-amino acid decarboxylase gene (eladocagene exuparvovec) and have completed 1-year evaluations. Rapid improvements in motor and cognitive function occurred within 12 months after gene therapy and were sustained during follow-up for >5 years. An increase in dopamine production was demonstrated by positron emission tomography and neurotransmitter analysis. Patient symptoms (mood, sweating, temperature, and oculogyric crises), patient growth, and patient caretaker quality of life improved. Although improvements were observed in all treated participants, younger age was associated with greater improvement. There were no treatment-associated brain injuries, and most adverse events were related to underlying disease. Post-surgery complications such as cerebrospinal fluid leakage were managed with standard of care. Most patients experienced mild to moderate dyskinesia that resolved in a few months. These observations suggest that eladocagene exuparvovec treatment for aromatic L-amino acid decarboxylase deficiency provides durable and meaningful benefits with a favorable safety profile.

Gene therapy in the putamen for curing AADC deficiency and Parkinson's disease.

This commentary provides an overview of the putamen as an established target site for gene therapy in treating aromatic l-amino acid decarboxylase (AADC) deficiency and Parkinson's disease, two debilitating neurological disorders that involve motor dysfunction caused by dopamine deficiencies. The neuroanatomy and the function of the putamen in motor control provide good rationales for targeting this brain structure. Additionally, the efficacy and safety of intraputaminal gene therapy demonstrate that restoration of dopamine synthesis in the putamen by using low doses of adeno-associated viral vector serotype 2 to deliver the hAADC gene is well tolerated. This restoration leads to sustained improvements in motor and nonmotor symptoms of AADC deficiency and improved uptake and conversion of exogenous l-DOPA into dopamine in Parkinson's patients.

Gene therapy for aromatic L-amino acid decarboxylase deficiency by MR-guided direct delivery of AAV2-AADC to midbrain dopaminergic neurons.

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare genetic disorder characterized by deficient synthesis of dopamine and serotonin. It presents in early infancy, and causes severe developmental disability and lifelong motor, behavioral, and autonomic symptoms including oculogyric crises (OGC), sleep disorder, and mood disturbance. We investigated the safety and efficacy of delivery of a viral vector expressing AADC (AAV2-hAADC) to the midbrain in children with AADC deficiency (ClinicalTrials.gov Identifier NCT02852213). Seven (7) children, aged 4-9 years underwent convection-enhanced delivery (CED) of AAV2-hAADC to the bilateral substantia nigra (SN) and ventral tegmental area (VTA) (total infusion volume: 80 µL per hemisphere) in 2 dose cohorts: 1.3 × 1011 vg (n = 3), and 4.2 × 1011 vg (n = 4). Primary aims were to demonstrate the safety of the procedure and document biomarker evidence of restoration of brain AADC activity. Secondary aims were to assess clinical improvement in symptoms and motor function. Direct bilateral infusion of AAV2-hAADC was safe, well-tolerated and achieved target coverage of 98% and 70% of the SN and VTA, respectively. Dopamine metabolism was increased in all subjects and FDOPA uptake was enhanced within the midbrain and the striatum. OGC resolved completely in 6 of 7 subjects by Month 3 post-surgery. Twelve (12) months after surgery, 6/7 subjects gained normal head control and 4/7 could sit independently. At 18 months, 2 subjects could walk with 2-hand support. Both the primary and secondary endpoints of the study were met. Midbrain gene delivery in children with AADC deficiency is feasible and safe, and leads to clinical improvements in symptoms and motor function.

A Machine Learning Approach to Differentiate Two Specific Breast Cancer Subtypes Using Androgen Receptor Pathway Genes.

Triple-negative breast cancer is a heterogeneous disease with different molecular and histological subtypes. The Androgen receptor is expressed in a portion of triple-negative breast cancer cases and the activation of the androgen receptor pathway is thought to be a molecular subtyping signature as well as a therapeutic target for triple-negative breast cancer. Thus, identification of the androgen receptor pathway status is important for both molecular characterization andclinical management. In this study, we investigate the expression of the androgen receptor pathway in metaplastic breast cancer and luminal androgen receptor subtypes of triple-negative breast cancer and found that the androgen receptor pathway was downregulated in metaplastic breast cancer compared to luminal androgen receptor subtype. Using random forest, we found that the two subtypes of breast cancer can be molecularly classified with the gene expression of the androgen receptor pathway.

Alteration of L-Dopa decarboxylase expression in SARS-CoV-2 infection and its association with the interferon-inducible ACE2 isoform.

L-Dopa decarboxylase (DDC) is the most significantly co-expressed gene with ACE2, which encodes for the SARS-CoV-2 receptor angiotensin-converting enzyme 2 and the interferon-inducible truncated isoform dACE2. Our group previously showed the importance of DDC in viral infections. We hereby aimed to investigate DDC expression in COVID-19 patients and cultured SARS-CoV-2-infected cells, also in association with ACE2 and dACE2. We concurrently evaluated the expression of the viral infection- and interferon-stimulated gene ISG56 and the immune-modulatory, hypoxia-regulated gene EPO. Viral load and mRNA levels of DDC, ACE2, dACE2, ISG56 and EPO were quantified by RT-qPCR in nasopharyngeal swab samples from COVID-19 patients, showing no or mild symptoms, and from non-infected individuals. Samples from influenza-infected patients were analyzed in comparison. SARS-CoV-2-mediated effects in host gene expression were validated in cultured virus-permissive epithelial cells. We found substantially higher gene expression of DDC in COVID-19 patients (7.6-fold; p = 1.2e-13) but not in influenza-infected ones, compared to non-infected subjects. dACE2 was more elevated (2.9-fold; p = 1.02e-16) than ACE2 (1.7-fold; p = 0.0005) in SARS-CoV-2-infected individuals. ISG56 (2.5-fold; p = 3.01e-6) and EPO (2.6-fold; p = 2.1e-13) were also increased. Detected differences were not attributed to enrichment of specific cell populations in nasopharyngeal tissue. While SARS-CoV-2 virus load was positively associated with ACE2 expression (r≥0.8, p<0.001), it negatively correlated with DDC, dACE2 (r≤-0.7, p<0.001) and EPO (r≤-0.5, p<0.05). Moreover, a statistically significant correlation between DDC and dACE2 expression was observed in nasopharyngeal swab and whole blood samples of both COVID-19 and non-infected individuals (r≥0.7). In VeroE6 cells, SARS-CoV-2 negatively affected DDC, ACE2, dACE2 and EPO mRNA levels, and induced cell death, while ISG56 was enhanced at early hours post-infection. Thus, the regulation of DDC, dACE2 and EPO expression in the SARS-CoV-2-infected nasopharyngeal tissue is possibly related with an orchestrated antiviral response of the infected host as the virus suppresses these genes to favor its propagation.

Next-generation sequencing reveals alternative L-DOPA decarboxylase (DDC) splice variants bearing novel exons, in human hepatocellular and lung cancer cells.

The human L-DOPA decarboxylase (DDC) is an enzyme that displays a pivotal role in metabolic processes. It is implicated in various human disorders, including hepatocellular and lung cancer. Several splice variants of DDC have previously been described, most of which encode for protein isoforms of this enzyme. In the present study, we used next-generation sequencing (NGS) technology along with nested touchdown PCR and Sanger sequencing to identify new splice variants bearing novel exons of the DDC gene, in hepatocellular and lung cancer cell lines. Using an in-house-developed algorithm, we discovered seven novel DDC exons. Next, we determined the structure of ten novel DDC transcripts, three of which contain an open reading frame (ORF) and probably encode for three previously unknown protein isoforms of this enzyme. Future studies should focus on the elucidation of their role in cellular physiology and cancer pathobiology.

L-Dopa-Decarboxylase (DDC) Is a Positive Prognosticator for Breast Cancer Patients and Epinephrine Regulates Breast Cancer Cell (MCF7 and T47D) Growth In Vitro According to Their Different Expression of Gi- Protein- Coupled Receptors.

A coherence between thyroid dysfunction and breast cancer incidence exists. Thyroid hormone metabolites bind to TAAR1 (trace amine-associated receptor 1) and through that modulate the serotonergic and dopaminergic system. Catecholamines themselves are synthesized by the L-dopa decarboxylase (DDC). The aim of our study was to analyze the influence of catecholamines on the DDC expression in primary breast cancer patients and the role of DDC concerning overall survival (OS). DDC expression was analyzed by immunohistochemistry. The effect of epinephrine on the expression of DDC and the Gi- protein was analyzed on the protein level via Western blot. A viability assay was performed to test the metabolic cell viability. The overexpression of DDC in the primary tumor was associated with longer OS (p = 0.03). Stimulation with epinephrine induced the downregulation of DDC (p = 0.038) and significantly increased viability in T47D cells (p = 0.028). In contrast, epinephrine induced an upregulation of DDC and decreased the proliferation of MCF7 cells (p = 0.028). Epinephrine led to an upregulation of Gi protein expression in MCF7 cells (p = 0.008). DDC is a positive prognostic factor for OS in breast cancer patients, and it is regulated through epinephrine differently in MCF7 and T47D. DDC may represent a novel target for the treatment of breast cancer, especially concerning its interaction with epinephrine.