Automatic segmentation of Nucleus Accumbens

Segmentation of subcortical structures in the brain has become an increasingly important topic in contemporary medicine. The ability to effi ciently isolate different regions of the human brain has allowed doctors and technicians to become more e fficient in the diagnosis of mental disorders and the evaluation of the patient conditions. An area of the brain whose possible segmentation has received particular attention is the Nucleus Accumbens, which is believed to play a central role in the reward circuit. In fact, studies of volumetric brain magnetic resonance imaging (MRI) have shown neuroanatomical abnormalities of this structure in adult attention defficit/hyperactivity disorder (ADHD), and speci cally a smaller average volume of the region. The use of a reliable automated segmentation method would therefore represent an extremely helpful and e fficient tool for identifying this disorder, especially when compared to manual volume labeling methods, which often turn out to be tedious and extremely time-consuming. However, automatic segmentation of the Accumbens is extremely di fficult to obtain, due to the lack of contrast with the surrounding structures. This means that most conventional segmentation methods are useless for this purpose, and makes the segmentation method selection a very delicate procedure. Consequently, the main objective of the thesis is the implementation of a robust algorithm for segmenting the Nucleus Accumbens structure. The research project aims to apply pre-existing segmentation methods to the Nucleus Accumbens, moving then to an evaluation of such methods and an estimation of how e ffective they are. Diff erent segmentation methods were used for this purpose; firstly, the standard Atlas Segmentation Approach was used, showing generally poor results paired with long computational times and high complexity. Moreover, this method has shown potential problems in the individuation of the correct region, leading, in some cases, to completely wrong segmentations. In addition to the fi rst method, Multi Atlas Segmentation and Adaptive Multi Atlas Segmentation methods have been implemented. The results have shown improved accuracy and better performance than the original method. Judging by the results, the segmentation of the Nucleus Accumbens has proven to be an extremely complicated task, both for the dimension of the structure itself and for the lack of contrast with the surrounding structures. In order to improve detection accuracy, combination of multiple methods is necessary, as using a single method for the segmentation process can lead to an incorrect labeling

Automatic segmentation of Nucleus Accumbens

Segmentation of subcortical structures in the brain has become an increasingly important topic in contemporary medicine. The ability to effi ciently isolate different regions of the human brain has allowed doctors and technicians to become more e fficient in the diagnosis of mental disorders and the evaluation of the patient conditions. An area of the brain whose possible segmentation has received particular attention is the Nucleus Accumbens, which is believed to play a central role in the reward circuit. In fact, studies of volumetric brain magnetic resonance imaging (MRI) have shown neuroanatomical abnormalities of this structure in adult attention defficit/hyperactivity disorder (ADHD), and speci cally a smaller average volume of the region. The use of a reliable automated segmentation method would therefore represent an extremely helpful and e fficient tool for identifying this disorder, especially when compared to manual volume labeling methods, which often turn out to be tedious and extremely time-consuming. However, automatic segmentation of the Accumbens is extremely di fficult to obtain, due to the lack of contrast with the surrounding structures. This means that most conventional segmentation methods are useless for this purpose, and makes the segmentation method selection a very delicate procedure. Consequently, the main objective of the thesis is the implementation of a robust algorithm for segmenting the Nucleus Accumbens structure. The research project aims to apply pre-existing segmentation methods to the Nucleus Accumbens, moving then to an evaluation of such methods and an estimation of how e ffective they are. Diff erent segmentation methods were used for this purpose; firstly, the standard Atlas Segmentation Approach was used, showing generally poor results paired with long computational times and high complexity. Moreover, this method has shown potential problems in the individuation of the correct region, leading, in some cases, to completely wrong segmentations. In addition to the fi rst method, Multi Atlas Segmentation and Adaptive Multi Atlas Segmentation methods have been implemented. The results have shown improved accuracy and better performance than the original method. Judging by the results, the segmentation of the Nucleus Accumbens has proven to be an extremely complicated task, both for the dimension of the structure itself and for the lack of contrast with the surrounding structures. In order to improve detection accuracy, combination of multiple methods is necessary, as using a single method for the segmentation process can lead to an incorrect labeling

Automatic segmentation of nucleus accumbens

Segmentation of subcortical structures in the brain through MRI scans has become an increasingly important topic in contemporary medicine. An area of the brain whose possible segmentation has received particular attention is the Nucleus Accumbens (NA), which is believed to play a central role in the reward circuit. The use of a reliable automated segmentation method would represent an extremely helpful and efficient tool for medical teams, since many disorders (such as ADHD) cause variations in the volume of the NA. Consequently, the main objective of the thesis is the implementation of a robust algorithm for segmenting the Nucleus Accumbens structure. We apply state of the art segmentation methods to the NA, using three different methods; firstly, the standard Atlas Segmentation Approach is used, showing generally poor results paired with long computational times and high complexity. In addition, Multi Atlas Segmentation and Adaptive Multi Atlas Segmentation methods are implemented. The results show improved accuracy and better performance than the single Atlas approach. Finally, we propose a prototype of GUI aimed at implementing a semi supervised segmentation approach, aimed at exploiting the knowledge of human experts to refine the segmentation results, and targeted to medical use

A Quantitative In Vitro Potency Assay for Adeno-Associated Virus Vectors Encoding for the UGT1A1 Transgene

Potency assessment of clinical-grade vector lots is crucial to support adeno-associated virus (AAV) vector release and is required for future marketing authorization. We have developed and validated a cell-based, quantitative potency assay that detects both transgenic expression and activity of an AAV8-hUGT1A1 vector, which is currently under clinical evaluation for the treatment of Crigler-Najjar syndrome. Potency of AAV8-hUGT1A1 was evaluated in vitro. After transduction of human hepatoma 7 (Huh7) cells, transgene-positive cells were quantified using flow cytometry and transgenic activity by a bilirubin conjugation assay. The in vitro potency of various AAV8-hUGT1A1 batches was compared with their potency in vivo. After AAV8-hUGT1A1 transduction, quantification of UGT1A1-expressing cells shows a linear dose-response relation (R2 = 0.98) with adequate intra-assay and inter-day reproducibility (coefficient of variation [CV] = 11.0% and 22.6%, respectively). In accordance, bilirubin conjugation shows a linear dose-response relation (R2 = 0.99) with adequate intra- and inter-day reproducibility in the low dose range (CV = 15.7% and 19.7%, respectively). Both in vitro potency assays reliably translate to in vivo efficacy of AAV8-hUGT1A1 vector lots. The described cell-based potency assay for AAV8-hUGT1A1 adequately determines transgenic UGT1A1 expression and activity, which is consistent with in vivo efficacy. This novel approach is suited for the determination of vector lot potency to support clinical-grade vector release

Efficacy of AAV8-hUGT1A1 with Rapamycin in neonatal, suckling, and juvenile rats to model treatment in pediatric CNs patients

A clinical trial using adeno-associated virus serotype 8 (AAV8)-human uridine diphosphate glucuronosyltransferase 1A1 (hUGT1A1) to treat inherited severe unconjugated hyperbilirubinemia (Crigler-Najjar syndrome) is ongoing, but preclinical data suggest that long-term efficacy in children is impaired due to loss of transgene expression upon hepatocyte proliferation in a growing liver. This study aims to determine at what age long-term efficacy can be obtained in the relevant animal model and whether immune modulation allows re-treatment using the same AAV vector. Neonatal, suckling, and juvenile Ugt1a1-deficient rats received a clinically relevant dose of AAV8-hUGT1A1, and serum bilirubin levels and anti-AAV8 neutralizing antibodies (NAbs) in serum were monitored. The possibility of preventing the immune response toward the vector was investigated using a rapamycin-based regimen with daily intraperitoneal (i.p.) injections starting 2 days before and ending 21 days after vector administration. In rats treated at postnatal day 1 (P1) or P14, the correction was (partially) lost after 12 weeks, whereas the correction was stable in rats injected at P28. Combining initial vector administration with the immune-suppressive regimen prevented induction of NAbs in female rats, allowing at least partially effective re-administration. Induction of NAbs upon re-injection could not be prevented, suggesting that this strategy will be ineffective in patients with low levels of preexisting anti-AAV NAbs

Prevalence and Relevance of Pre-Existing Anti-Adeno-Associated Virus Immunity in the Context of Gene Therapy for Crigler-Najjar Syndrome

Adeno-associated virus (AAV) vector-mediated gene therapy is currently evaluated as a potential treatment for Crigler-Najjar syndrome (CN) (NCT03466463). Pre-existing immunity to AAV is known to hinder gene transfer efficacy, restricting enrollment of seropositive subjects in ongoing clinical trials. We assessed the prevalence of anti-AAV serotype 8 (AAV8) neutralizing antibodies (NAbs) in subjects affected by CN and investigated the impact of low NAb titers (<1:5) on liver gene transfer efficacy in an in vivo passive immunization model. A total of 49 subjects with a confirmed molecular diagnosis of CN were included in an international multicenter study (NCT02302690). Pre-existing NAbs against AAV8 were detected in 30.6% (15/49) of screened patients and, in the majority of positive cases, cross-reactivity to AAV2 and AAV5 was detected. To investigate the impact of low NAbs on AAV vector-mediated liver transduction efficiency, adult wild-type C57BL/6 mice were passively immunized with pooled human donor-derived immunoglobulins to achieve titers of up to 1:3.16. After immunization, animals were injected with different AAV8 vector preparations. Hepatic vector gene copy number was unaffected by low anti-AAV8 NAb titers when column-purified AAV vector batches containing both full and empty capsids were used. In summary, although pre-existing anti-AAV8 immunity can be found in about a third of subjects affected by CN, low anti-AAV8 NAb titers are less likely to affect liver transduction efficiency when using AAV vector preparations manufactured to contain both full and empty capsids. These findings have implications for the design of liver gene transfer clinical trials and for the definition of inclusion criteria related to seropositivity of potential participants

Gene Therapy in Patients with the Crigler-Najjar Syndrome

Background: Patients with the Crigler-Najjar syndrome lack the enzyme uridine diphosphoglucuronate glucuronosyltransferase 1A1 (UGT1A1), the absence of which leads to severe unconjugated hyperbilirubinemia that can cause irreversible neurologic injury and death. Prolonged, daily phototherapy partially controls the jaundice, but the only definitive cure is liver transplantation. Methods: We report the results of the dose-escalation portion of a phase 1-2 study evaluating the safety and efficacy of a single intravenous infusion of an adeno-associated virus serotype 8 vector encoding UGT1A1 in patients with the Crigler-Najjar syndrome that was being treated with phototherapy. Five patients received a single infusion of the gene construct (GNT0003): two received 2×1012 vector genomes (vg) per kilogram of body weight, and three received 5×1012 vg per kilogram. The primary end points were measures of safety and efficacy; efficacy was defined as a serum bilirubin level of 300 μmol per liter or lower measured at 17 weeks, 1 week after discontinuation of phototherapy. Results: No serious adverse events were reported. The most common adverse events were headache and alterations in liver-enzyme levels. Alanine aminotransferase increased to levels above the upper limit of the normal range in four patients, a finding potentially related to an immune response against the infused vector; these patients were treated with a course of glucocorticoids. By week 16, serum bilirubin levels in patients who received the lower dose of GNT0003 exceeded 300 μmol per liter. The patients who received the higher dose had bilirubin levels below 300 μmol per liter in the absence of phototherapy at the end of follow-up (mean [±SD] baseline bilirubin level, 351±56 μmol per liter; mean level at the final follow-up visit [week 78 in two patients and week 80 in the other], 149±33 μmol per liter). Conclusions: No serious adverse events were reported in patients treated with the gene-therapy vector GNT0003 in this small study. Patients who received the higher dose had a decrease in bilirubin levels and were not receiving phototherapy at least 78 weeks after vector administration. (Funded by Genethon and others; ClinicalTrials.gov number, NCT03466463.)

Preclinical Development of an AAV8-hUGT1A1 Vector for the Treatment of Crigler-Najjar Syndrome

Adeno-associated viruses (AAVs) are among the most efficient vectors for liver gene therapy. Results obtained in the first hemophilia clinical trials demonstrated the long-term efficacy of this approach in humans, showing efficient targeting of hepatocytes with both self-complementary (sc) and single-stranded (ss) AAV vectors. However, to support clinical development of AAV-based gene therapies, efficient and scalable production processes are needed. In an effort to translate to the clinic an approach of AAV-mediated liver gene transfer to treat Crigler-Najjar (CN) syndrome, we developed an (ss)AAV8 vector carrying the human UDP-glucuronosyltransferase family 1-member A1 (hUGT1A1) transgene under the control of a liver-specific promoter. We compared our construct with similar (sc)AAV8 vectors expressing hUGT1A1, showing comparable potency in vitro and in vivo. Conversely, (ss)AAV8-hUGT1A1 vectors showed superior yields and product homogeneity compared with their (sc) counterpart. We then focused our efforts in the scale-up of a manufacturing process of the clinical product (ss)AAV8-hUGT1A1 based on the triple transfection of HEK293 cells grown in suspension. Large-scale production of this vector had characteristics identical to those of small-scale vectors produced in adherent cells. Preclinical studies in animal models of the disease and a good laboratory practice (GLP) toxicology-biodistribution study were also conducted using large-scale preparations of vectors. These studies demonstrated long-term safety and efficacy of gene transfer with (ss)AAV8-hUGT1A1 in relevant animal models of the disease, thus supporting the clinical translation of this gene therapy approach for the treatment of CN syndrome. Keywords: AAV vector, Crigler-Najjar syndrome, UGT1A1, liver gene transfer, long-term safet

Preclinical Development of an AAV8-hUGT1A1 Vector for the Treatment of Crigler-Najjar Syndrome

Adeno-associated viruses (AAVs) are among the most efficient vectors for liver gene therapy. Results obtained in the first hemophilia clinical trials demonstrated the long-term efficacy of this approach in humans, showing efficient targeting of hepatocytes with both self-complementary (sc) and single-stranded (ss) AAV vectors. However, to support clinical development of AAV-based gene therapies, efficient and scalable production processes are needed. In an effort to translate to the clinic an approach of AAV-mediated liver gene transfer to treat Crigler-Najjar (CN) syndrome, we developed an (ss)AAV8 vector carrying the human UDP-glucuronosyltransferase family 1-member A1 (hUGT1A1) transgene under the control of a liver-specific promoter. We compared our construct with similar (sc)AAV8 vectors expressing hUGT1A1, showing comparable potency in vitro and in vivo. Conversely, (ss)AAV8-hUGT1A1 vectors showed superior yields and product homogeneity compared with their (sc) counterpart. We then focused our efforts in the scale-up of a manufacturing process of the clinical product (ss)AAV8-hUGT1A1 based on the triple transfection of HEK293 cells grown in suspension. Large-scale production of this vector had characteristics identical to those of small-scale vectors produced in adherent cells. Preclinical studies in animal models of the disease and a good laboratory practice (GLP) toxicology-biodistribution study were also conducted using large-scale preparations of vectors. These studies demonstrated long-term safety and efficacy of gene transfer with (ss)AAV8-hUGT1A1 in relevant animal models of the disease, thus supporting the clinical translation of this gene therapy approach for the treatment of CN syndrome