Gene Therapy for Alan Handon Dudley Syndrome, and Methods and Formulations for the Combination of Gene Therapy and DITPA Therapy

Abstract

The present disclosure relates to a method for treating Allan-Herndon-Dudley syndrome, the method comprising administering 3,5-diiodothyropropionic acid (DITPA) to a subject in need thereof and performing gene therapy on the subject by introducing normal human MCT8 into the cells of the subject to increase T3 in the subject's brain.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 388,235, filed on Jul. 11, 2022, the entire content of which is incorporated herein by reference.

[0002] Government Licensing Rights This invention was made with government support under grant number DK15070 awarded by the National Institutes of Health (NIH). The government has certain rights in this invention.

[0003] The present invention relates to a method of gene therapy for treating a subject having Allan - Herndon - Dudley syndrome, and a method of combining administration of 3,5 - diiodothyropropionic acid (DITPA) to a subject having Allan - Herndon - Dudley syndrome with gene therapy administered to the subject.

Background Art

[0004] Allan - Herndon - Dudley syndrome ( "AHDS") is an X - linked recessive developmental disorder that causes intellectual and movement disorders in males. Specifically, AHDS patients have a mutated SLC16A2 gene, as a result of which the monocarboxylate transporter 8 ( "MCT8") protein is abnormal. MCT8 is a cell - membrane transporter for thyroid hormones.

[0005] In 2004, Allan Herndon and Dudley 1 demonstrated that the severe neuropsychomotor disorder observed in males in 1944 was caused by a defect in the MCT8 gene (also known as SLC16A2). 2 These people (mostly children) were unable to walk or talk due to a lack of the active thyroid hormone T3 in the brain. However, at the same time, because the excess T3 present in the blood entered other body tissues through another transporter, these children had increased metabolism and did not gain weight.

[0006] The symptoms of AHDS are usually caused by the lack of uptake of triiodothyronine ("T3"), a thyroid hormone transported across the cell membrane by MCT8, into cells. Due to this MCT8 deficiency, T3 is insufficient in the tissues necessary for it to function properly, and T3 accumulates in the serum. The other thyroid hormone, thyroxine ("T4"), usually remains at normal serum levels in AHDS patients, although it may decrease slightly from normal levels. In AHDS patients, thyroid-stimulating hormone ("TSH") is slightly elevated from normal.

[0007] When the thyroid hormone analog diiodothyropropionic acid (DITPA) was administered to MCT8-deficient mice, it had an inhibitory effect equivalent to that observed in normal mice on thyroid-stimulating hormone (TSH). 3 This suggested that DITPA could enter cells even in the absence of MCT8. When DITPA was compassionately administered to four MCT8-deficient infants, a decrease and normalization of elevated blood T3 were shown, so hypermetabolism and nutrition were improved, but neuropsychomotor disorders 4 were not improved.

[0008] When normal human MCT8 was introduced into cells of neonatal mice using the viral vector AAV9, T3 increased in the brains of neonatal Mct8-deficient mice, and T3-mediated effects could be induced. 5 However, these mice did not have nerve damage caused by another transporter. Recently, the same gene therapy was performed on prepubertal Mct8-deficient dKO mice with neurocognitive abnormalities. Neurological abnormalities, learning ability, and recall ability were improved, but elevated blood T3, which causes an increase in metabolism, was not improved. 6

[0009] Currently, there is no treatment for AHDS approved by the US Food and Drug Administration. Clinical trials of triiodothyroacetic acid ("TRIAC"), a drug used for the treatment of AHDS, have been completed. However, since TRIAC is structurally similar to T3, it becomes difficult to accurately evaluate T3 serum levels. Furthermore, TRIAC has been shown to significantly reduce T4 serum levels. See Groeneweg et al. Lancet Diabetes Endocrinol. 2019 Sep;7(9);695-706.

[0010] Combined treatment with a gene and DITPA should improve both neuropsychomotor and metabolic disorders that could not be achieved by each treatment alone and provide a complete recovery of the genetic defect.

Summary of the Invention

Problems to be Solved by the Invention

[0011] Alan H. Dudding syndrome ("AHDS") is an X-linked recessive developmental disorder that causes intellectual and motor disabilities in males. Specifically, AHDS patients have a mutated SLC16A2 gene, as a result of which the monocarboxylate transporter 8 ("MCT8") protein is abnormal. The symptoms of AHDS are usually caused by the lack of uptake of triiodothyronine ("T3"), a thyroid hormone that is normally transported across the cell membrane by MCT8, into cells. This MCT8 deficiency results in a lack of T3 in the tissues necessary for T3 to function properly, and T3 accumulates in the serum. The other thyroid hormone, thyroxine ("T4"), usually remains at normal serum levels in AHDS patients, although it may decrease slightly from normal levels. In AHDS patients, thyroid-stimulating hormone ("TSH") is slightly elevated from normal.

[0012] 3,5-Diiodothyropropionic acid ("DITPA") is another thyroid hormone analog being studied for the treatment of AHDS. However, as noted above, DITPA is still not approved for use in the treatment of AHDS. This lack of approval may be due to the absence of an effective dosing regimen, a stable and effective composition, and extensive pharmacological evaluation. International Publication No. WO 2012 / 171065, published on December 20, 2012, attempts to establish a DITPA dosing regimen for AHDS patients, but this publication provides only theoretical examples.

[0013] When compassionate administration of DITPA was given to four MCT8-deficient infants, a decrease and normalization of elevated blood T3 were shown, so hypermetabolism and nutrition were improved, but neuropsychomotor disorders 4 were not improved.

[0014] When normal human MCT8 was introduced into cells using the viral vector AAV9 in neonatal mice, T3 increased in the brains of neonatal Mct8-deficient mice, and T3-mediated effects could be induced 5 . However, these mice did not have neuropathy caused by another transporter. Recently, the same gene therapy was performed on prepubertal Mct8-deficient dKO mice with neurocognitive abnormalities. Neurological abnormalities, learning ability, and recall ability were improved, but elevated blood T3 that causes an increase in metabolism was not improved 6 .

[0015] Combined therapy with the gene and DITPA should improve both neuropsychomotor and metabolic disorders that could not be achieved by each treatment alone and provide complete recovery of the genetic defect. Therefore, there is a need for gene therapy effective for the treatment of AHDS and the symptoms of AHDS, particularly gene therapy combined with a DITPA dosing regimen.

[0016] References 1. Allan W, Herndon CN, Dudley FC. Some examples of the inheritance of mental deficiency: apparently sex-linked idiocy and microcephaly. Am J Ment Defic. 1944;48:325 - 334. 2. Dumitrescu AM, Liao XH, Best TB, Brockmann K, Refetoff S. A Novel Syndrome Combining Thyroid and Neurological Abnormalities Is Associated with Mutations in a Monocarboxylate Transporter Gene. Am J Hum Genet. 2004;74(1):168 - 175. 3. Di Cosmo C, Liao XH, Dumitrescu AM, Weiss RE, Refetoff S. A thyroid hormone analogue with reduced dependence on the monocarboxylate transporter 8 (MCT8) for tissue transport. Endocrinology. 2009;150(9):4450 - 4458. 4. Verge CF, Konrad D, Cohen M, et al. Diiodothyropropionic Acid (DITPA) in the Treatment of MCT8 Deficiency. J Clin Endocrinol Metab. 2012;97(12):4515 - 4523. 5. Iwayama H, Liao XH, Braun L, et al. Adeno Associated Virus 9-Based Gene Therapy Delivers a Functional Monocarboxylate Transporter 8, Improving Thyroid Hormone Availability to the Brain of Mct8-Deficient Mice. Thyroid. 2016;26(9):1311-1319. 6. Liao X-H, P.A, Shelest O, et al. AAV9-MCT8 delivery at juvenile stage ameliorates neurological and behavioral deficits in an Allan-Herndon-Dudley Syndrome mouse model. 2021.

Means for Solving the Problem

[0017] Disclosure The present subject matter relates to a method of gene therapy for treating a subject having Allan-Herndon-Dudley syndrome and a method of combining such gene therapy with the administration of 3,5-diiodothyropropionic acid (DITPA) for treating a subject having Allan-Herndon-Dudley syndrome.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0019] I. Gene Therapy Background: Allan-Herndon-Dudley syndrome (AHDS) is a severe psychomotor disorder that also exhibits characteristic abnormalities in thyroid hormone (TH) levels. AHDS is caused by inactivating mutations in the monocarboxylate transporter 8 (MCT8), a specific TH cell membrane transporter that is widely expressed in the central nervous system (CNS). MCT8 mutations cause impaired transport of TH across the blood-brain barrier, leading to insufficient TH supply to the nerves. Currently, there is no successful treatment for the neurological symptoms. Previous studies have shown that intravenous administration of adeno-associated virus serotype 9 (AAV9)-based gene therapy to neonatal Mct8 knockout (Mct8- / y) male mice, rather than intracerebroventricularly, increased brain T3 content and partially restored TH-dependent gene expression, suggesting a promising approach for treating this neuropathy.

[0020] Methods: Feasibility of intravenous delivery of AAV9 carrying human MCT8 A well-established Mct8- / y / organic anion transporting polypeptide 1c1 (Oatp1c1)- / - double knockout (dKO) mouse model of AHDS was tested, which, unlike MCT8- / y mice, displays both neurological and TH phenotypes. Furthermore, because this condition is usually diagnosed in childhood, P30 mice were given intravenous treatment and blinded psychomotor testing was performed at P120–P140, after which tissues were harvested and analyzed.

[0021] RESULTS: Systemic intravenous delivery of AAV9-MCT8 during juvenile stages led to improved motor and cognitive function at P120-P140, accompanied by near normalization of T3 content and increased responses of positively regulated TH-dependent gene expression in different brain regions examined (thalamus, hippocampus, and parietal cortex). Effects on serum TH concentrations and peripheral tissues were less pronounced, showing only improvements in serum T3 / rT3 ratio and hepatic deiodinase 1 expression.

[0022] Conclusion: Intravenous administration of AAV9 carrying human MCT8 to young dKO mice with AHDS has long-term beneficial effects, primarily on the CNS. This preclinical study demonstrates that this gene therapy improves the severe neurological symptoms of AHDS patients.

[0023] In one embodiment, the present technology relates to a method of treating and ameliorating neurological abnormalities associated with AHDS in a subject in need of such treatment using gene therapy, the gene therapy comprising administration of AAV9-MCT8 to the subject, the administration being preferably intravenous.

[0024] introduction Thyroid hormone (TH) is essential for the development and metabolic homeostasis of most organs and tissues (1). The main form of TH released from the thyroid into the bloodstream is thyroxine (T4), which acts as a prohormone. The conversion of T4 to the active hormone triiodothyronine (T3) or to the inactive reverse T3 (rT3) occurs intracellularly by iodothyronine deiodinases (2). The main mechanism of T3 action is achieved by binding to specific nuclear receptors, which function as regulators of gene transcription (3). Since the metabolism and action of TH are intracellular events, the presence of TH-specific transporters that mediate the uptake and efflux of TH by cells is required (4). The solute carrier family 16, member 2 (SLC16A2) gene, located on the X chromosome, encodes the monocarboxylate transporter 8 (MCT8) protein (5). MCT8 is well conserved throughout vertebrate evolution and is widely expressed in the body and the central nervous system (CNS) (6). An important function of MCT8 is to facilitate the transport of TH across the cell membrane (5).

[0025] Inactivating mutations in the MCT8 gene in males cause severe psychomotor retardation (7 - 9) and are clinically described as Allan-Herndon-Dudley syndrome (AHDS) (10). Patients exhibit neurological disorders including severe intellectual disability, decreased trunk muscle tone, dystonia, and movement disorders. MCT8 deficiency also causes a TH phenotype that includes elevated serum T3 levels, decreased rT3 and T4, and normal or slightly elevated thyroid-stimulating hormone (TSH), resulting in a marked increase in the free T3 / T4 and T3 / rT3 ratios (11).

[0026] Two independently generated Mct8-KO mouse models (12, 13) closely recapitulate the TH phenotype observed in AHDS patients but do not show the expected neurological or behavioral phenotypes. This is because mild TH deficiency occurs in the mouse brain due to a T4-specific transporter that does not exist in the human blood-brain barrier (BBB). Specifically, organic anion transporting polypeptide 1c1 (Oatp1c1), encoded by the slco1c1 gene, was identified in mouse brain capillaries but not in human brain capillaries (14-16). Double knockout mct8 - / y , oatp1c1 - / - mice (dKO) exhibit disease-related phenotypes including impaired TH transport into the CNS, resulting in a significant decrease in the number of cortical parvalbumin-positive GABAergic interneurons, decreased myelination, and prominent motor abnormalities (17). These results indicate that in mice, Mct8 (along with Oatp1c1) plays an important role in the transport of TH into the CNS and, importantly, provides a powerful disease model for human MCT8 deficiency (18, 19). To progress from animal models to human-based models, induced pluripotent stem cells (iPSCs) were derived from AHDS patients and differentiated into brain microvascular endothelial cells, which showed MCT8-dependent transport of TH across the human BBB (16, 20). However, MCT8 is not limited to brain endothelium and also affects TH transport across the cell membranes of neurons (21).

[0027] Gene therapy offers a promising approach for treating single-gene diseases. Patients with spinal muscular atrophy type 1, who have a deleterious mutation in the SMN1 gene, were treated by a single intravenous injection of adeno-associated virus serotype 9 (AAV9) containing DNA encoding SMN, resulting in improved survival and achievement of motor milestones and motor function (22, 23), and subsequently, the ZOLGENSMA® gene therapy product received FDA approval. Additional examples of beneficial gene therapy approaches for single-gene diseases have been recently reported (24-26).

[0028] Intracerebroventricular (ICV) delivery targets the ventricles directly, thereby bypassing the blood-brain barrier (BBB), while intravenous (IV) delivery provides systemic delivery and mainly transduces tissues outside the CNS including blood vessels. Importantly, IV delivery of AAV9 has been shown to cross the BBB and efficiently infect CNS cells (27). In a recent proof-of-concept study, an AAV9-MCT8 construct was delivered to neonatal Mct8-KO (MCT8 - / y ) mice by ICV or IV injection (28), and TH signaling in the brain increased with IV delivery but not with ICV delivery. However, since no neuropathy has been shown in Mct8-KO mice, it is unclear whether this approach will lead to the alleviation of neurological symptoms.

[0029] Here, the inventors tested the potential of IV delivery of AAV9-MCT8 in dKO mice. The inventors chose to treat young male mice at postnatal day 30 (P30) and tested the potential recovery of adult neurological and behavioral parameters. This approach was supported because the diagnosis of MCT8 deficiency is usually made in childhood.

[0030] Materials and Methods All procedures were approved by the Institutional Animal Care and Use Committee (IACUC #009128) of Cedars-Sinai Medical Center. Female and male mct8 - / - / oatp1c1 - / - mice with a C57BL / 6 background and mct8 - / y / oatp1c1 - / -Male mice were paired to generate dKO offspring. WT C57BL / 6 was used as a control. Only male mice were selected for all treatments. AAV9-MCT8 containing 50×10¹⁰ viral particles vp / g in a volume of 20 μl / g was administered to P30 juvenile dKO mice and controls via tail vein injection. Behavioral measurements, as well as biochemical and molecular measurements, were all performed on tissues and sera at P120 - P140 and analyzed in a double-blind manner without knowing which group the mice belonged to. The identity of the mice was not known to those administering the AAV9-MCT8 and was not known to the technicians during the behavioral assays, dissections, tissue collections, and biochemical analyses. This was not blinded during the collection of results and statistical analysis. The animals received behavioral analysis before being sacrificed for tissue collection. Therefore, results from the same mice are shown in all figures.

[0031] Statistical analysis All scatter plots were first tested for normal distribution using the Kolmogorov - Smirnov test with Dallal - Wilkinson - Lilliefor for corrected P - values. Normally distributed datasets were tested using one - way ANOVA with Tukey's post - hoc test for multiple comparisons. Non - normally distributed datasets were compared using the Mann - Whitney non - parametric test for independent samples. Behavioral data collected over time, including the rotarod test and Barnes maze, were analyzed by mixed - model regression using random intercepts and fixed factors of time (as a continuous variable), treatment group, and the interaction term of group and time. To compare learning curves between groups, β - coefficients were compared, and a difference was considered significant at a step - down Bonferroni - corrected alpha level of <0.05. Residuals were examined to confirm the goodness - of - fit of the model. Data analysis was performed using GraphPad Prism v8.0.0 and SAS Enterprise Guide v8.2. All data are presented as mean ± SEM. p < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0032] Result IV delivery of AAV9-MCT8 at P30 improves the motor ability of dKO mice.

[0033] Considering that AHDS patients are often diagnosed in childhood, the feasibility of treatment needs to be optimally tested in infancy. In previous experience (28), IV is a simpler route and has been shown to be more feasible than ICV delivery as it targets not only the brain but also other body regions expressing MCT8 such as the liver. Therefore, the inventors tested IV delivery of 50 × 1010 vp / g of AAV9-MCT8 (28), a previously tested dose, to prepubertal P30 dKO mice (Figure 1A).

[0034] To confirm the expression of human MCT8 by viral delivery, the liver and brain were harvested at P140. As expected, human MCT8 mRNA expression was not detected in wild-type (WT) or untreated dKO mice (data not shown). MCT8 expression was observed in the liver and various brain regions of AAV9-MCT8 IV-treated dKO mice, with the highest levels seen in the liver. Higher MCT8 levels were shown in the pituitary and choroid plexus compared to the BBB-protected regions of the thalamus, parietal cortex, and hippocampus (Figure 1B).

[0035] Mice were subjected to various behavioral analyses to evaluate motor function, which were performed at P120 where differences between WT and dKO had been previously reported (29). In the evaluation by the rotarod test, untreated dKO mice showed an overall shortening of the latency to fall and a shortening of the learning curve compared to the WT group (Figure 2A). After IV delivery of AAV9-MCT8, dKO mice showed a significant prolongation of the latency to fall and an increase in the learning curve, indicating that treatment improved motor ability and potentially cognitive function. Neither the overall motor activity nor the rearing behavior evaluated by the open field test was significantly different in untreated dKO mice compared to WT mice, and there was no significant change after treatment (Figures 2B and 2C). Finally, in the analysis of the hindlimbs, there was no significant improvement in stride length, and it was shown that the hindlimb angle was fully recovered (Figures 2D and 2E). Collectively, the data indicate that AAV9-MCT8 treatment at P30 restores some, but not all, of the motor parameters in dKO mice.

[0036] IV delivery of AAV9-MCT8 at P30 improves the cognitive ability of dKO mice.

[0037] Learning, spatial memory, and memory recall were evaluated using the Barnes maze (30). During the 4-day training period, treatment did not significantly improve the learning curve (Figure 3A). After a 2-day rest, untreated dKO mice required a longer latency compared to untreated WT mice and treated dKO mice (Figure 3B). Next, the position of the escape hole was moved during the 2-day training, but no significant differences were observed among all groups during this period (Figure 3C). These data suggest that IV treatment of dKO mice with AAV9-MCT8 at P30 partially restored learning and recall abilities.

[0038] The spontaneous alternation (Y-maze) test was used to further examine spatial memory. Untreated dKO mice showed a significant decrease in the rate of spontaneous alternation compared to untreated WT mice, but there was no significant improvement in dKO mice treated with AAV9-MCT8 (Figure 3D). The results of both tests suggest that IV treatment of P30 dKO mice with AAV9-MCT8 improves cognitive ability and partially restores spatial and learning memory.

[0039] IV delivery of AAV9-MCT8 at P30 partially restores T3 content in the brains of dKO mice.

[0040] To evaluate the impact on pathophysiology, serum, liver, and brain were collected at P140 (Figure 1A). Examination of brain T3 content showed that T3 levels in the thalamus (Figure 4A) and hippocampus (Figure 4B) of treated dKO mice were completely normalized and reached WT levels. A significant increase in brain T3 content was also observed in the frontal cortex (Figure 4C). Considering the low level of MCT8 expression in the brain after treatment, these results suggest that low MCT8 expression (Figure 1B) is sufficient to normalize brain T3 content.

[0041] IV delivery of AAV9-MCT8 at P30 corrects T3-induced gene expression.

[0042] To evaluate the effects of T3 in different brain regions, we next studied T3-induced gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). These genes were selected based on known responses to T3 (31). The hairless (Hr) levels were significantly improved in the hypothalamus (Figure 4D), hippocampus (Figure 4E), and frontal cortex (Figure 4F) of treated dKO mice. Aldehyde dehydrogenase 1 family member a1 (Aldh1a1) levels were fully restored in the hypothalamus (Figure 4G) and hippocampus (Figure 4H) and significantly improved in the frontal cortex (Figure 4I). Finally, carbonyl reductase 2 (Cbr2) levels were also significantly improved in treated dKO mice compared to untreated dKO mice in the hypothalamus, hippocampus, and frontal cortex (Figures 4J–L). These results suggest that IV delivery of AAV9-MCT8 to P30 dKO mice substantially improves brain T3 content and T3-induced gene expression and can be maintained long term.

[0043] IV delivery of AAV9-MCT8 at P30 partially restores the liver phenotype and minimally restores the serum phenotype in dKO mice.

[0044] In IV delivery, the MCT8 expression level in the liver is expected to be significantly increased (Figure 1B), and it can easily penetrate into the blood vessels of the liver compared to the BBB capillaries (32). In contrast to TH deficiency in the brain, AHDS patients experience TH excess in peripheral tissues due to high serum T3 levels. Therefore, the inventors measured the T3 level in the liver (Figure 5A). This treatment did not result in a significant decrease in T3 levels in the treated dKO mice. Analysis of liver T3-induced genes was performed by qRT-PCR. The expression levels of deiodinase 1 (Dio1), malic enzyme 1 (Me1), and uncoupling protein 2 (Ucp2) (Figure 5B - D), as well as the liver T3 level, were all significantly increased in the untreated dKO group compared to WT littermates, confirming the effect of TH excess in the liver. By AAV9-MCT8 delivery, the Dio1 mRNA level in the treated dKO mice was significantly decreased compared to untreated dKO mice. However, there was no significant decrease in the levels of Me1 and Ucp2 in response to the treatment.

[0045] AHDS patients have abnormal serum TH levels with elevated T3, decreased rT3 and T4, and normal or slightly elevated TSH, resulting in lower ratios of T3 / T4 and T3 / rT3. Increased hepatic deiodinase 1 enzyme activity is one of the mechanisms underlying these serum thyroid tests, and a decrease in its expression is required to improve this phenotype (33). To test the effect of IV delivery of AAV9-MCT8 at P30 on the serum TH phenotype in dKO mice, blood was collected from P140 mice and serum TSH and TH levels were quantified. The serum levels of TSH, T4, T3, and rT3, as well as the T3 / rT3 and T3 / T4 ratios, all changed significantly in dKO mice compared to WT littermates (Figure 5E - J). AAV9-MCT8 treatment did not significantly change the serum levels of TSH, T4, T3, rT3, and the T3 / T4 ratio, but the combination of a slight decrease in T3 and an increase in rT3 resulted in a significant decrease in the T3 / rT3 ratio, consistent with the observed attenuation of Dio1 expression. This indicates that delivering AAV9-MCT8 in infancy can partially improve serum abnormalities.

[0046] Discussion In this study, we tested the possibility of intravenous (IV) delivery of AAV9-MCT8 to young dKO mice. Our analysis showed long-term expression of human MCT8 in the central nervous system (CNS) and peripheral tissues, suggesting that AAV9 can efficiently transduce cells with MCT8 in peripheral tissues and the CNS. Restoration of MCT8 expression not only improved motor and cognitive behaviors but also significantly restored T3 content and related gene expression in different regions of the brain.

[0047] Patients with MCT8 deficiency suffer from severe neuropsychomotor phenotypes and TH excess in peripheral tissues. Therefore, effective treatment strategies need to consider the insufficient transport of TH across the blood-brain barrier and neuronal plasma membranes and the excess of TH in peripheral tissues. Since AHDS is a rare disease, it is often misdiagnosed, and as a result, the disease is identified later. Furthermore, currently, there are approximately 300 diagnosed cases, most of which are older children (11, 34). Therefore, it is important to develop effective treatments even in juveniles. Thus, we tested the effect of tail vein IV delivery at P30 around puberty, when pathophysiological symptoms are evident in both dKO mice and patients (17, 35, 36).

[0048] Endogenously, MCT8 is ubiquitously expressed and prominently localized in the thyroid, liver, kidney, and CNS (6, 12, 13). In this study, we showed that IV administration of AAV9-MCT8 to P30 dKO mice induced long-term MCT8 expression in the CNS and liver.

[0049] In the brain, MCT8 expression was observed in various regions, and it was confirmed that the AAV9 vector can pass through the blood-brain barrier and efficiently transduce brain cells during this developmental period (27). Interestingly, higher expression was observed in brain regions not protected by the blood-brain barrier, such as the choroid plexus and pituitary gland, compared to the thalamus, hippocampus, and cortex (37, 38). Surprisingly, in this study, this long-term brain expression almost completely normalized the brain T3 concentration and related gene expression in the thalamus, hippocampus, and parietal cortex. MCT8 deficiency has previously been suggested to be caused by both reduced T3 transport across the blood-brain barrier and reduced T3 transport across the neuronal membrane (16, 21, 29, 39). Therefore, these results suggest that restoration of brain T3 content has been achieved. However, future studies are needed to distinguish whether the improvement in brain content is caused by MCT8 expression in cerebral blood vessels or the choroid plexus (both of which function as entrances to the brain).

[0050] Improvement in brain content also enhanced the performance of the rotarod test and gait analysis. The reason why other motor functions did not change significantly remains unclear, but the observed improvement is thought to be due to improvement in psychomotor function. The treated animals also showed an improvement in the learning curve using the rotarod test, suggesting that the treatment may have beneficial effects on cognitive and motor functions. Exploration, learning, and memory are thought to be derived from the hippocampus (40). Notably, a slight recovery of hippocampus-dependent learning and memory in the Barnes maze test in response to treatment was observed to correlate with a significant increase in T3 levels and T3-induced gene expression in the hippocampus.

[0051] MCT8 expression was significantly higher in the liver than in the brain. However, no significant recovery was observed in liver T3 levels. Nevertheless, a decrease in liver Dio1 expression, a TH-regulatory enzyme that generates T3 from T4 and contributes to part of the T3 excess in serum, was observed (33). This effect on Dio1 expression partially improved the serum T3 / rT3 ratio, but other parameters were not significantly improved. These results suggest a modest beneficial effect of systemic (IV) delivery of MCT8 to peripheral tissues. Additional mechanisms, such as decreased thyroid secretion and changes in negative feedback to the hypothalamus and pituitary, contribute to the characteristic serum thyroid tests in AHDS, and thus the availability of TH is different (41 - 43). Therefore, to enhance partial recovery, additional TH normalization treatments need to be considered in combination with gene therapy.

[0052] Currently, there are several treatment strategies focused on TH analogs. These thyroid-mimicking compounds need to activate the TH-induced transcriptional pathway via thyroid nuclear receptors and need to cross the cell membrane independently of MCT8. In animal models, TH analogs such as DITPA (44, 45), TRIAC (18), TETRAC (19), and sovetilome (46) can restore some of the peripheral and central abnormalities, but their effects on neurological symptoms were limited or remained unclear because mice without neurological impairment were used. In AHDS patients, DITPA (47) and TRIAC (48) decreased the high T3 concentration in serum, but there was no evidence of improvement in neurological symptoms. Therefore, TH analogs are thought to be used in combination with gene therapy.

[0053] Chemical and pharmaceutical chaperones have also been proposed as alternative approaches. These chaperones can restore the ability of some MCT8 gene mutations to transport TH across the cell membrane in animal models (49, 50), but their clinical effects have not been evaluated to date. Furthermore, the use of chemical chaperones is limited to only a few missense mutations and thus cannot be applied to the majority of patients.

[0054] Gene therapy, which has emerged as a promising approach for treating monogenic developmental neuropathies (23, 25, 26, 51 - 53), may overcome these limitations and has the potential to target all mutations and patients. Furthermore, restoration of functional MCT8 may also resolve unconfirmed roles of MCT8, such as the transport of additional potential substrates, in addition to TH transport.

[0055] This study indicates a promising therapeutic direction but also has limitations. dKO mice provide a useful model of AHDS, but the symptoms are milder than those in patients. Furthermore, features associated with additional diseases, such as lack of speech or a tendency to death, could not be addressed in this study due to the limitations of the model.

[0056] Overall, this study shows that intravenous administration of AAV9 - MCT8 to dKO mice provides substantial restoration of molecular and biochemical parameters in the brain and also improves the TH over - effect in peripheral tissues. Furthermore, this treatment improves motor and behavioral abilities. These findings support future clinical trials of AAV - based MCT8 gene therapy in AHDS patients.

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Allan W, Herndon CN, Dudley FC 1944 Some examples of the inheritance of mental deficiency: apparently sex - linked idiocy and microcephaly. Am J Ment Defic 46:325 - 334 11. Groeneweg S, van Geest FS, Abaci A, Alcantud A, Ambegaonkar GP, Armour CM, Bakhtiani P, Barca D, Bertini ES, van Beynum IM et al. 2020 Disease characteristics of MCT8 deficiency: an international, retrospective, multicentre cohort study. Lancet Diabetes Endocrinol 8:594 - 605 12. Dumitrescu AM, Liao XH, Weiss RE, Millen K and Refetoff S. 2006 Tissue-specific thyroid hormone deprivation and excess in monocarboxylate transporter (Mct)8-deficient mice. Endocrinology 147:4036-4043 13. Trajkovic M, Visser TJ, Mittag J, Horn S, Lukas J, Darras VM, Raivich G, Bauer K and Heuer H. 2007 Abnormal thyroid hormone metabolism in mice lacking the monocarboxylate transporter 8. J Clin Invest 117:627-635 14. Mayerl S, Visser TJ, Darras VM, Horn S and Heuer H. 2012 Impact of Oatp1c1 deficiency on thyroid hormone metabolism and action in the mouse brain. Endocrinology 153:1528-1537 15. Roberts LM, Woodford K, Zhou M, Black DS, Haggerty JE, Tate EH, Grindstaff KK, Mengesha W, Raman C and Zerangue N. 2008 Expression of the thyroid hormone transporters monocarboxylate transporter-8 (SLC16A2) and organic ion transporter-14 (SLCO1C1) at the blood-brain barrier. Endocrinology 149:6251-6261 16. Vatine GD, Al-Ahmad A, Barriga BK, Svendsen S, Salim A, Garcia L, Garcia VJ, Ho R, Yucer N, Qian T, Lim RG, Wu J, Thompson LM, Spivia WR, Chen Z, Van Eyk J, Palecek SP, Refetoff S, Shusta EV and Svendsen CN. 2017 Modeling Psychomotor Retardation using iPSCs from MCT8-Deficient Patients Indicates a Prominent Role for the Blood-Brain Barrier. Cell Stem Cell 20:831-843 e5 17. 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Anbalagan S, Gordon L, Blechman J, Matsuoka RL, Rajamannar P, Wircer E, Biran J, Reuveny A, Leshkowitz D, Stainier DYR, Levkowitz G. 2018 Pituicyte Cues Regulate the Development of Permeable Neuro-Vascular Interfaces. Dev Cell 47:711-726 e5 38. Ben-Zvi A, Liebner S 2021 Developmental regulation of barrier- and non-barrier blood vessels in the CNS. J Intern Med https: / / doi.org / 10.1111 / joim.13263. 39. Ceballos A, Belinchon MM, Sanchez-Mendoza E, Grijota-Martinez C, Dumitrescu AM, Refetoff S, Morte B and Bernal J. 2009 Importance of monocarboxylate transporter 8 for the blood-brain barrier-dependent availability of 3,5,3’-triiodo-L-thyronine. Endocrinology 150:2491-2496 40. Voss JL, Gonsalves BD, Federmeier KD, Tranel D and Cohen NJ. 2011 Hippocampal brain-network coordination during volitional exploratory behavior enhances learning. Nat Neurosci 14:115-120 41. Trajkovic-Arsic M, Visser TJ, Darras VM, Friesema EC, Schlott B, Mittag J, Bauer K and Heuer H. 2010 Consequences of monocarboxylate transporter 8 deficiency for renal transport and metabolism of thyroid hormones in mice. Endocrinology 151:802-9 42. Di Cosmo C, Liao XH, Dumitrescu AM, Philp NJ, Weiss RE, Refetoff S. 2010 Mice deficient in MCT8 reveal a mechanism regulating thyroid hormone secretion. J Clin Invest 120: 3377-88 43. Fliers E, Unmehopa UA, Alkemade A 2006 Functional neuroanatomy of thyroid hormone feedback in the human hypothalamus and pituitary gland. Mol Cell Endocrinol. 151:1-8 44. Di Cosmo C, Liao XH, Dumitrescu AM, Weiss RE and Refetoff S. 2009 A thyroid hormone analog with reduced dependence on the monocarboxylate transporter 8 for tissue transport. Endocrinology 150:4450-4458 45. Ferrara AM, Liao XH, Ye H, Weiss RE, Dumitrescu AM and Refetoff S. 2015 The thyroid hormone analog DITPA ameliorates metabolic parameters of male mice with Mct8 deficiency. Endocrinology 156:3889-3894 46. Barez-Lopez S, Hartley MD, Grijota-Martinez C, Scanlan TS and Guadano-Ferraz A. 2018 Sobetirome and its Amide Prodrug Sob-AM2 Exert Thyromimetic Actions in Mct8-Deficient Brain. Thyroid 28:1211-1220 47. Verge CF, Konrad D, Cohen M, Di Cosmo C, Dumitrescu AM, Marcinkowski T, Hameed S, Hamilton J, Weiss RE, Refetoff S. 2012 Diiodothyropropionic acid (DITPA) in the treatment of MCT8 deficiency. J Clin Endocrinol Metab 97:4515 - 4523 48. Groeneweg S, Peeters RP, Moran C, et al. 2019 Effectiveness and safety of the triiodothyronine analogue Triac in children and adults with MCT8 deficiency: an international, single - arm, open - label, phase 2 trial. Lancet Diabetes Endocrinol 7:695 - 706 49. Braun D, Schweizer U 2017 The chemical chaperone phenylbutyrate rescues MCT8 mutations associated with milder phenotypes in patients with Allan - Herndon - Dudley syndrome. Endocrinology 158:678 - 691 50. Braun D, Schweizer U 2015 Efficient activation of pathogenic Δphe501 mutation in monocarboxylate transporter 8 by chemical and pharmacological chaperones. Endocrinology 156:4720 - 30 51. Mendell JR, Al-Zaidy S, Shell R, et al. 2017 Single-dose gene-replacement therapy for spinal muscular atrophy. N Engl J Med 377:1713-1722 52. White KA, Nelvagal HR, Poole TA, Lu B, Johnson TB, Davis S, Pratt MA, Brudvig J, Assis AB, Likhite S, Meyer K, Kaspar BK, Cooper JD, Wang S and Weimer JM. 2021 Intracranial delivery of AAV9 gene therapy partially prevents retinal degeneration and visual deficits in CLN6-Batten disease mice. Mol Ther-Methods Clin Dev 20:497-507. 53. Pearson TS, Gupta N, San Sebastian W, et al. 2021 Gene therapy for aromatic Lamino acid decarboxylase deficiency by MR-guided direct delivery of AAV2-AADC to midbrain dopaminergic neurons. Nat Commun 12:4251.

[0058] II. DITPA Administration The applicant discovered the administration timing of 3,5-diiodothyropropionic acid (DITPA), which is surprisingly effective in treating Alan Houghton Dudley syndrome (AHDS).

[0059] In one embodiment, the present technology relates to a method for treating AHDS, the method comprising orally administering DITPA to a pregnant mother who has chosen to carry an affected male embryo twice a day, or orally administering it three times a day to a subject with MCT8 deficiency who needs it to reduce and normalize high blood T3 levels and improve hypermetabolism and nutrition. Administration to pregnant women is initiated within 11 weeks after conception of the subject, preferably between 8 and 10 weeks after conception. The dosage of DITPA for a pregnant mother to treat an affected male infant is 0.5 ± 0.2 mg per day, and the postnatal dosage for affected infants and children of any age is 1.5 ± 0.5 mg per day.

[0060] In another embodiment, the present technology relates to a method for treating Allan-Herndon-Dudley syndrome, the method comprising a) administering DITPA daily for two weeks to a subject who needs it at a first dosage; b) administering DITPA daily for two weeks to the subject at a second dosage greater than the first dosage; c) measuring the serum level of triiodothyronine (“T3”) in the subject, and if the T3 serum level is normal, the second dosage is administered daily; d) optionally, adjusting the daily dosage of DITPA administered to the subject based on the T3 serum level of the subject measured in step c), wherein if the T3 serum level is too high, a third dosage is administered daily, the third dosage being greater than the second dosage, and if the T3 serum level is too low, a fourth dosage is administered daily, the fourth dosage being less than the second dosage; e) optionally, measuring the T3 serum level of the subject on approximately the 28th day after the first administration of the third or fourth dosage, and if the T3 serum level is normal, the third or fourth dosage is administered daily; f) Optionally, based on the T3 serum level of the subject measured in step e), adjusting the daily dose of DITPA administered to the subject, wherein if the serum T3 level is too low after the daily administration of the third dose, the second dose is administered to the subject, if the serum T3 level is too low after the daily administration of the fourth dose, the subject is administered daily at the first dose, and if the serum T3 level is too high after the daily administration of the fourth dose, the second dose is administered daily to the subject, and including.

[0061] In a preferred embodiment, the first dose is about 1 milligram per kilogram of the subject's body weight per day ("mg / kg / day").

[0062] In another preferred embodiment, the second dose is about 2 mg / kg / day.

[0063] In another preferred embodiment, the third dose is about 2.5 mg / kg / day.

[0064] In another preferred embodiment, the fourth dose is about 1.5 mg / kg / day.

[0065] As used herein, the term "too high" refers to a T3 serum level that is about 15% higher than the T3 serum level considered normal for the subject's age.

[0066] As used herein, the term "too low" refers to a T3 serum level that is 15% lower than the T3 serum level considered normal for the subject's age.

[0067] As used herein, the "normal" T3 serum levels by age of the subject are disclosed in Lem et al., Serum thyroid hormone levels in healthy children from birth to adulthood and in short children born small for gestational age, J Clin Endocrinol Metab, 2012 Sep, 97(9), 3170 - 8, doi:10.1210 / jc.2012 - 1759, Epub 2012 Jun 26.

[0068] In another embodiment, the present disclosure relates to a method of treating AHDS, the method comprising: a) administering DITPA to a subject in need thereof daily for 2 weeks at a first dosage; and b) administering DITPA to the subject daily for 2 weeks at a second dosage that is more than the first dosage, wherein the daily administration starts on the 3rd day after the subject's birth.

[0069] In a preferred embodiment, the daily dosage of DITPA is administered once a day to a subject in need thereof; more preferably, the daily dosage of DITPA is divided into two parts, each part being administered every 12 hours; most preferably, the daily dosage of DITPA is divided into three parts, each part being administered every 8 hours.

[0070] In a preferred embodiment, the administration of DITPA is via the oral route.

[0071] In one embodiment, DITPA can be formulated in a composition comprising DITPA or a salt thereof and one or more pharmaceutically acceptable excipients.

[0072] In a preferred embodiment, DITPA or a salt thereof can be present in the pharmaceutical composition of the present subject matter at a concentration of about 0.001% to about 10% w / w or w / v.

[0073] In a preferred embodiment, one or more pharmaceutically acceptable excipients may be present in the pharmaceutical composition of the present disclosure at a concentration of about 90% to about 99.999% w / w or w / v.

[0074] Pharmaceutically acceptable excipients suitable for use in the subject matter of the present invention include, but are not limited to, disintegrants, binders, fillers, plasticizers, lubricants, penetration enhancers, surfactants, sweeteners, sweetening enhancers, flavoring agents, and pH adjusters.

[0075] As used herein, the term "disintegrant" refers to a pharmaceutically acceptable excipient that promotes the disintegration of a tablet when the tablet comes into contact with water or other liquids. Disintegrants suitable for use in the present technology include, but are not limited to, natural starches such as corn starch and potato starch, directly compressible starches such as starch 1500, modified starches such as carboxymethyl starch, sodium hydroxymethyl starch, and sodium starch glycolate, as well as starch derivatives such as amylose, cross-linked polyvinylpyrrolidone such as crospovidone, cross-linked sodium carboxymethyl cellulose, sodium hydroxymethyl cellulose, calcium hydroxymethyl cellulose, croscarmellose sodium, modified celluloses such as low-substituted hydroxypropyl cellulose, alginic acid, sodium alginate, microcrystalline cellulose, methacrylic acid divinylbenzene copolymer salts, and combinations thereof.

[0076] Binders suitable for use in the present technology include, but are not limited to, polyethylene glycol, soluble hydroxyalkyl cellulose, polyvinylpyrrolidone, gelatin, natural rubber, and combinations thereof.

[0077] Fillers suitable for use in the present technology include, but are not limited to, dibasic calcium phosphate, tribasic calcium phosphate, anhydrous calcium hydrogen phosphate, calcium sulfate and dicalcium sulfate, lactose, sucrose, amylose, dextrose, mannitol, inositol, and combinations thereof.

[0078] Plasticizers suitable for use in the present subject matter include, but are not limited to, microcrystalline cellulose, triethyl citrate, polyhexanediol, acetylated monoglyceride, glyceryl triacetate, castor oil, and combinations thereof.

[0079] Lubricants suitable for use in the present technology include, but are not limited to, magnesium stearate, sodium stearyl fumarate, stearic acid, glyceryl behenate, micronized polyethylene glycol, talc, anhydrous colloidal silica, and combinations thereof.

[0080] Penetration enhancers suitable for use in the present subject matter include, but are not limited to, precipitated silica, maltodextrin, β-cyclodextrin, menthol, limonene, carvone, methyl chitosan, polysorbate, sodium lauryl sulfate, glyceryl oleate, caproic acid, enanthic acid, pelargonic acid, capric acid, undecylenic acid, lauric acid, myristic acid, palmitic acid, oleic acid, stearic acid, linolenic acid, arachidonic acid, benzethonium chloride, benzethonium bromide, benzalkonium chloride, cetylpyridinium chloride, disodium edetate dihydrate, sodium deoxycholate, sodium deoxyglycolate, sodium glycolate, sodium caprate, sodium taurocholate, sodium hydroxybenzoyl aminocaprylate, dodecyldimethylaminopropionate, L-lysine, glyceryl oleate, glyceryl monostearate, citric acid, peppermint oil, and combinations thereof.

[0081] Surfactants suitable for use in the present subject matter include, but are not limited to, sorbitan esters, sodium docecylate, sodium lauryl sulfate, cetrimide, and combinations thereof.

[0082] Sweeteners suitable for use in the present technology include, but are not limited to, aspartame, saccharin, acesulfame potassium, sodium saccharinate, neohesperidin dihydrochalcone, sucralose, sucrose, dextrose, mannitol, glycerin, xylitol, and combinations thereof.

[0083] Sweetness enhancers suitable for use in the present technology include, but are not limited to, the ammonium salt forms of crude and purified glycyrrhizic acid.

[0084] Flavoring agents suitable for use in the present subject include, but are not limited to, peppermint oil, menthol, spearmint oil, citrus oil, cinnamon oil, strawberry flavor, cherry flavor, raspberry flavor, orange oil, tutti-frutti flavor, and combinations thereof.

[0085] pH adjusters suitable for use in the present formulation include, but are not limited to, hydrochloric acid, citric acid, fumaric acid, lactic acid, sodium hydroxide, sodium citrate, sodium bicarbonate, sodium carbonate, ammonium carbonate, sodium acetate, and combinations thereof.

[0086] In another preferred embodiment, the pharmaceutical composition of the present technology does not contain a preservative.

[0087] The pharmaceutical composition of the present technology can be formulated in any dosage form including, but not limited to, measured, aerosol containing powder and spray, chewable bar, beads, coated, film-coated, gel-coated capsules, liquid-filled and coated pellets, cell sheets, chewable gels, concentrates, elixirs, emulsions, films containing solubility, films for solutions and films for suspensions, gels containing measured gels, small balls, granules containing granules for solutions, granules for suspensions, chewing gums, inhalants, injections containing foams, liposomes, emulsions, lipid complexes, powders, lyophilized powders and liposome suspensions, liquids, medicinal drops, ointments, patches, electrically controlled patches, pellets, implantable pellets, pills, powders, powders, measured powders, solutions, measured solutions, solution concentrates, gel-forming solutions / solution drops, sprays, measured sprays, suspensions, suspensions, syrups, tablets, chewable tablets, coated tablets, coated particles in tablets, film-coated tablets, tablets for solutions, tablets for suspensions, orally disintegrating tablets, soluble tablets, sugar-coated tablets, dispersible tablets, tablets with sensors, tapes, troches and wafers, and their sustained-release and delayed-release forms.

[0088] In a preferred embodiment, the pharmaceutical composition of the present technology is in the form of a tablet. In a more preferred embodiment, the pharmaceutical composition of the present formulation is in the form of a dispersible tablet. In an even more preferred embodiment, the pharmaceutical composition of the present formulation is in the form of a water-dispersible tablet. In the most preferred embodiment, the pharmaceutical composition of the present formulation is in the form of a water-dispersible tablet, and the tablet is scored so that it can be divided into four equal parts.

[0089] In a preferred embodiment, when the pharmaceutical composition of the present technology is in the form of a water-dispersible tablet, the dispersion time of the tablet is about 70 seconds or less, more preferably about 60 seconds or less, even more preferably about 40 seconds or less, even more preferably about 30 seconds or less, even more preferably about 20 seconds or less, even more preferably about 10 seconds or less, and even more preferably about 5 seconds or less.

[0090] As used herein, the term "pharmaceutically acceptable" refers to components that are not biologically or otherwise undesirable in oral applications.

[0091] As used herein, all numerical values regarding amounts, weights, etc. are defined as their respective specific values "about", i.e., plus or minus 10%. For example, the phrase "10% w / w" should be understood as "9% - 11% w / w". Accordingly, amounts within 10% of the claimed value are included within the scope of the claim.

[0092] As used herein, "% w / w" refers to the weight percentage of the entire formulation.

[0093] As used herein, "% w / v" refers to the weight percentage based on the volume of the entire formulation.

[0094] As used herein, the term "effective amount" refers to the amount necessary to treat a subject in need of treatment.

[0095] As used herein, the term "treatment" or "treating" refers to alleviating or improving AHDS or the symptoms of AHDS.

[0096] As used herein, the term "stable" includes, but is not limited to, physical and chemical stability.

[0097] Pharmaceutically acceptable salts that can be used in accordance with this subject matter include, but are not limited to, hydrochloride, hydrochloride dihydrate, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, hydrogen tartrate, ascorbate, succinate, mesylate, maleate, gentisinate, fumarate, tannate, sulfate, tosylate, esylate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoic acid)) salts.

[0098] Throughout this application, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" include plural references.

[0099] The disclosed embodiments are merely exemplary embodiments of the inventive concepts disclosed herein and should not be considered limiting unless explicitly stated otherwise in the claims.

[0100] III. Combination of Gene Therapy and DITPA Administration Administration of DITPA may be used to lower and normalize elevated blood T3 and improve hypermetabolism and nutrition, but does not improve the neuropsychomotor disorders associated with AHDS. The use of gene therapy helps to improve neurological abnormalities, learning ability, and recall ability, but does not help to improve elevated blood T3 that causes increased metabolism. Combining DITPA treatment and gene therapy improves both the neuropsychomotor and metabolic disorders that could not be achieved by each treatment alone, and the genetic defect is completely restored.

[0101] In one embodiment, the present technology relates to a method of using a combination of DITPA administration and gene therapy to a subject to treat and improve neurological and metabolic abnormalities associated with AHDS in the subject in need thereof, the gene therapy comprising administration of AAV9-MCT8 to the subject, and DITPA being administered daily to reduce and normalize elevated blood T3 and improve hypermetabolism and nutrition in the subject, and preferably, AAV9-MCT8 is administered intravenously to the subject.

[0102] The following examples are intended to illustrate the present technology and to teach those skilled in the art how to use the formulations of this new technology. They are not intended to be limiting in any way.

Example

[0103] Example 1 - Administration Plan (Predictive) for Prenatal Subjects Method DITPA was administered to the mother of a prenatal male subject who had previously tested positive for the SLC16A2 allele correlated with Allan-Herndon-Dudley syndrome, starting at 4 weeks after conception and ending at the subject's birth, in three divided doses at 8-hour intervals at a daily dose of 1 mg / kg / day.

[0104] Results This administration plan was successful in reducing the symptoms of AHDS in the neonatal subject compared to affected neonates whose mothers did not receive treatment with DITPA.

[0105] Example 2 - In Vitro Evidence of the Direct Effect of SRW-101 (DITPA) on the Reduction of T3 Generated from T4: SRW101 (DITPA) Inhibits D1 Enzyme Activity in the Liver In Vitro DITPA reduces the activity of iodothyronine deiodinase 1 in the liver in vivo and in vitro (see the dose-response of DITPA added to the liver in vitro and the measurement of D1 enzyme activity, i.e., the conversion from T4 to T3). This is the main mechanism for the reduction of T3 and the increase of T4 by reducing consumption. This has been shown to occur in humans with MCT8 deficiency.

[0106] These are the results of the normalization of serum T3 levels, which are important endocrine biomarkers and parameters of treatment effect, to measure the expected metabolic changes resulting from the normalization of thyroid examinations.

[0107] More specifically, it is expected that by reducing T3, which acts on peripheral tissues to promote metabolism, the ability to improve nutrition and increase body weight will be enhanced.

[0108] Important measurements such as weight gain (age-corrected) and metabolic parameters (cholesterol, creatine kinase, SHBG) are secondary evaluation items.

[0109] Observations with annotations by parents, such as sleep, diet records, and exercise activities, are very valuable.

[0110] In vitro measurement of the dose response of DITPA added to the liver and D1 enzyme activity (conversion from T4 to T3) Figure 6 shows in vitro evidence of the direct effect of DITPA, which reduces T3 generated from T4 rather than reducing T3 by reducing T4 by TSH suppression as in the case of TRIAC. T4 is important for the brain even in the presence of reduced uptake due to MCT8 deficiency. The data in Figure 6 reflect the effect of adding DITPA to the liver (in vitro) as measured by D1 enzyme activity, i.e., the conversion from T4 to T3.

[0111] Example 3 - Background understanding regarding thyroid physiology There are three objectives in the research centered on gene expression control and gene therapy.

[0112] (1) To elucidate the mechanisms that generate thyroid phenotypes in which several newly identified genetic defects are observed. These include the selenoenzymes deiodinases D1 and D3, PKHD1L1 mutations from the study of Pkdh1l1KO mice, and LRP2 mutations by in vitro structural and functional characterization.

[0113] (2) To elucidate the mechanism of resistance to TSH (RTSH) caused by mutations in primate-specific short tandem repeats (STRs) on chromosome 15. The recently co-developed human thyroid organoids are used to generate STR mutant thyroid organoids using CRISPR / Cas9 or PiggyBac transposons as genome editing tools to study the physiological function of this primate-specific STR and its role in the dominant genetic phenotype of RTSH. The TSH sensitivity of normal and mutant organoids is measured in vitro or in vivo after transplantation into hypothyroid mice.

[0114] (3) To determine the efficacy of combination therapy of genes and thyroid hormone (TH) analogs in monocarboxylic acid 8 (MCT8) deficiency. MCT8 deficiency associated with the X chromosome causes a disease known as Alan Houghton Dudley syndrome (AHDS) in male infants, accompanied by severe neuropsychomotor disorders caused by defects in TH transport in the brain and systemic thyrotoxicosis caused by excess circulating T3. Double knockout (dKO) mice lacking Mct8 and the TH transporter Oatp1c1 recapitulate the findings of AHDS.

[0115] The present inventors recently showed that gene therapy of pre-pubertal dKO mice using adeno-associated virus 9 (AAV9) containing human MCT8 cDNA normalized the T3 content in the brain and improved motor and cognitive functions, but could not improve serum thyroid tests. Adding diiodothyropropionic acid (DITPA) or triiodothyroacetic acid (TRIAC), TH analogs known to correct the thyrotoxicosis of AHDS in peripheral tissues but not the neuropsychomotor symptoms, is restoring the relief of this debilitating disease.

[0116] Specific objectives Goal #3. To determine the efficacy of combination gene therapy and TH analog therapy in MCT8 deficiency. Theoretical basis and implications: Deficiency of X-linked MCT8 causes a complex phenotype in male infants, including severe neuropsychomotor disorders caused by deficiency of TH transport in the brain and systemic thyrotoxicosis caused by excess circulating T3. Treatment with the TH analogues diiodothypropionic acid (DITPA) or triiodothyroacetic acid (TRIAC) improves thyrotoxicosis in peripheral tissues but does not improve neuropsychomotor disorders. Double knockout (dKO) mice deficient in Mct8 and the TH transporter Oatp1c1 recapitulate the findings of AHDS. The inventors have recently shown that gene therapy of prepubertal dKO mice using adeno-associated virus 9 (AAV9) containing human MCT8 cDNA normalized brain T3 content and improved motor and cognitive functions, but could not improve serum TFT. Combination therapy with AAV9 and TH analogues may restore MCT8 deficiency and function as a preclinical model for human treatment.

[0117] The inventors do the following: 1. Treat dKO mice in combination with AAV9-hMCT8 alone and the TH analogues DITPA and TRIAC. Serum TFT and the tissue content of T3, T4 and analogues, as well as the expression of TH-responsive genes, are evaluated in different brain regions and organs.

[0118] In addition to motor function, the effects on metabolism and the heart are also evaluated using a metabolic cage.

[0119] Research strategy Importance The inventors have identified a defect in the monocarboxylate 8 (MCT8), a TH transmembrane transporter that causes severe neuropsychomotor syndrome [Allan-Herndon-Dudley syndrome (AHDS)], 6 as well as defects in TH metabolism, SBP2 7 and DIO1 8 and discovered.

[0120] The inventors have investigated a highly important area, namely, (i) expanding the study of newly identified genetic defects that affect thyroid physiology, including deiodinases, and (ii) developing potentially effective treatments for AHDS. Preliminary data indicate that this proposal is clinically and scientifically relevant and underscores the strength of its translation.

[0121] Another important aspect is the development of treatments for AHDS. Those who have seen children affected by AHDS will immediately appreciate the urgent need for effective treatments. Currently, the use of TH analogs that only improve hypermetabolism is the focus, but the inventors have recently developed a promising gene therapy strategy. Our compelling new data show that administration of adeno-associated virus 9 (AAV9) expressing human MCT8 cDNA improves the neuropsychomotor component of AHDS even when administered around puberty. 15 Gene therapy in combination with TH analogs holds great promise for long-term improvement of the overall phenotype of AHDS.

[0122] Innovation AHDS 17 The treatment of patients remains a major challenge in thyrology. The inventors propose a pioneering approach in the field of thyroidology that uses AAV9-mediated transduction of normal human MCT8 cDNA to deliver functional hMCT8 to a mouse model of AHDS (mice lacking Mct8 and Oatp1c1) during infancy, an approach that partially restores neurokinetic abnormalities 15 while simultaneously using TH analog therapy to improve hypermetabolism. 18、19 This combined approach targets the complex pathophysiology of MCT8 deficiency and may restore or reduce the severe phenotype of this deficiency and serve as a preclinical model for treatment in humans.

[0123] Objective 3. Determine the efficacy of the combination of gene therapy and TH analog therapy in MCT8 deficiency. MCT8 deficiency causing severe neurological symptoms due to impaired TH transport in the brain and thyrotoxicosis in other tissues 6、24 Since its discovery, multiple patients have been identified. However, treatment remains difficult. Although the hypermetabolic state was improved by the use of TH analogs, neuropsychomotor symptoms could not be improved. The inventors have shown that when an adeno-associated virus serotype 9 vector expressing human MCT8 cDNA (AAV9-hMCT8) is intravenously (IV) administered to 1-day-old Mct8KO mice, hMCT8 protein is distributed in the choroid plexus. Their brain T3 content increased, and the expression of genes regulated by TH 25 also increased. Injection into 30-day-old Mct8 and Oatp1c1-deficient (dKO) mice showing thyroid and neurological abnormalities resulted in improved motor ability, learning ability, and recall ability 15 . This was accompanied by an increase in brain T3 content and the expression of genes regulated by TH 15 . However, few changes were observed in serum and peripheral tissues. The inventors are currently proposing to combine gene therapy and TH analogs to improve both hypermetabolism and neuropsychomotor disorders 26 .

[0124] Example 5 - Gene Therapy Specific objective #3. Determine the efficacy of combined gene and TH analog therapy in MCT8 deficiency Background: MCT8 deficiency is a syndrome with two elements: severe neurodevelopmental delay with gait disturbance, dystonia, poor head control, and mental retardation, and a characteristic pattern of thyroid test abnormalities including elevated serum T3 and TSH and decreased T4 and rT3 6、24 . This syndrome was previously described as X-linked mental retardation in males by Alan, Haldon, and Dudley in 1944 60 and is now called AHDS, but it was later found to be caused by mutations in the MCT8 gene 61 .

[0125] Mouse models of Mct8 deficiency reproduce human thyroid test abnormalities 62、63However, since Oatp1c1, another TH cell membrane transporter that is present in very small amounts in the human brain, is expressed in the brain, they do not show neurological abnormalities. 64 Indeed, mice lacking both Mct8 and Oatp1, or double KO (dKO), exhibit both thyroid and neurokinetic disorders. 65 。

[0126] It is now clear that selective brain TH deficiency is caused by a significant reduction in hormone transport at the level of vascular endothelial cells or the blood-brain barrier (BBB). 66、67 Thus, MCT8 deficiency causes TH deficiency in the brain, causes neurokinetic abnormalities, and TH excess due to increased circulating T3 reaching tissues by alternative transporters causes cardiotoxicity and hypermetabolism. Thus, in contrast to TH deficiency caused by congenital thyroid deficiency or defects in TH synthesis, the combination of selective tissue deficiency and excessive MCT8 deficiency cannot be treated with TH replacement. 68 。

[0127] For this purpose, it has been proposed to devise new therapies that affect both elements of the deficiency. This requires a combination of treatments to improve both hypermetabolism and neuropsychomotor deficits, with the goal of changing the natural history of the deficiency 26 by improving mobility, reducing complications, extending survival, and promoting long-term care.

[0128] Rationale: Studies in Mct8-deficient mice have shown that the TH analogs diiodothyropropionic acid (DITPA) or triiodothyroacetic acid (TRIAC) enter cells independently of MCT8. 69、70 Trials using these analogs in children with MCT8 deficiency reduced high serum T3 levels, decreased hypermetabolism, decreased heart rate, and improved several parameters reflecting the action of TH on peripheral tissues. However, they had no significant effect on neuropsychomotor disorders 19、71 and no changes in neurodevelopment were recorded with long-term use of TRIAC. 18 。

[0129] The inventors requested gene therapy using an adeno-associated virus serotype 9 (AAV9) vector, which has been shown to restore spinal muscular atrophy 72 and has received FDA approval. The inventors showed that when AAV9 carrying human MCT8 cDNA was administered intravenously to 1-day-old Mct8-deficient mice, the presence of hMCT8 protein in the choroid plexus increased the brain's T3 content and the expression of genes positively regulated by TH 25 . Injection into 30-day-old dKO mice showing thyroid and neurological abnormalities resulted in improved motor, learning, and recall abilities 15 . This was accompanied by increased T3 content and TH-regulated gene expression in all three brain regions examined, namely the male thalamus, hippocampus, and parietal cortex (Figure 7) 15 .

[0130] However, despite significant expression of hMCT8 in this tissue, little change was observed in liver markers and no change was observed in serum TH concentrations except for a minimal decrease in the T3 / rT3 ratio 15 . These initial results of gene therapy are very promising because a single treatment has a lasting effect (over 100 days) on brain function in a mouse model very similar to human AHDS. More importantly, it is still effective when applied at P30. Therefore, to further improve potential gene therapy, the inventors propose combining it with a treatment that can reduce serum T3 concentration.

[0131] Experimental approach: The proposed protocol is outlined schematically in Figure 8.

[0132] 50×10 10Inject AAV9-hMCT8 containing virus particles / g into the tail vein of P30 dKO mice. WT mice are given the same amount of empty virus vector (AAV9 without hMCT8). Three days later, a group of 10 animals is treated with DITPA and TRIAC at doses that normalize the serum T3 concentration in dKO mice. These are administered by daily intraperitoneal injection and are 0.3 mg of DITPA and 6 μg of TRIAC per 100 g of body weight. (1) Obtain baseline measurements of serum T4, T3, rT3, TSH, cholesterol, alkaline phosphatase (AP), and creatine kinase (CK) the day before. Repeat the same tests after 10 days (P40), adjusting the doses of DITPA and TRIAC according to the serum levels of T3 while maintaining T4 at normal levels of 3.5 - 5.0 μg / dL in adult C57Bl / 6J male mice.

[0133] Collect another blood sample at P60 and again at P120. (2) Conduct behavioral and motor tests (rotarod, open field, gait analysis, Barnes maze, Y maze) over a 20-day period. At the same time intervals, while not undergoing behavioral and motor tests, perform metabolic studies using indirect calorimetry in a metabolic cage as previously done in the laboratory 73 to evaluate the metabolic and cardiac effects of combination therapy. Measure total energy expenditure (TEE), respiratory exchange ratio (RER), total activity, food and water intake, and heart rate. Continuously measure physical activity and also measure O2 uptake and CO2 production at 30-minute intervals. RER, TEE, glucose, and lipid oxidation are calculated from O2 consumption (VO2) and CO2 production (VCO2) relative to body weight.

[0134] In P140, after collection of peripheral blood samples, the animals are perfused under anesthesia prior to tissue collection. Tissue from two animals per group is used for immunohistochemistry and confocal microscopy to identify the location of hMCT8 protein. Tissues are collected as fragments of whole brain, anterior pituitary, thyroid, liver, kidney, heart, and muscle (gastrocnemius), and immediately frozen. Subsequently, the frozen brain is dissected to recover frontal, parietal, and occipital cortex, hippocampus, thalamus, hypothalamus, striatum, choroid plexus, and cerebellum. Tissues are analyzed for the content of T3, T4, and their respective analogs (DITPA and TRIAC), as well as the expression of tissue-specific TH regulatory genes and the enzyme activities of the three deiodinases.

[0135] The expression of the following genes is measured by qPCR. In the brain (Hr, Dio2, Dio3, Aldh1a1, Cbr2), in the pituitary (Tshb, Gh, Trhr, Dio2), in the thyroid (Tshr, Tg, Tpo, Duox2, Nis, Ttf1), in the liver (Dio1, Usp2, Me1), in the heart (Serca2, Myh6, Myh7), in the muscle (Hr, Myh1, 2 and 7, Mct4, Pkm), in the kidney (Dio1, NaPi2, Clc2). The effects on other transporters (Mct10, Lat1, Lat2) and the injected human MCT8 are also measured. Some monoclonal antibodies can measure T3 without interference from DITPA 74 but it should be noted that this is not the case for TRIAC 75 . Therefore, T3 is measured by liquid chromatography-tandem mass spectrometry (LC-MS / MS) with DITPA and TRIAC as required 45 .

[0136] Expected results, potential difficulties, additional experiments, alternatives: Based on this information, the following effects are expected. (a) DITPA should normalize all three iodothyronines by reducing D1 activity, as observed in vivo 19 73, this effect has been demonstrated in vitro (Figure 6). Thus, as demonstrated by the change in the marker of TH action, it reduces hypermetabolism, and (b) TRIAC should reduce serum T3, but is thought to be reduced by reducing serum T4 through a central mechanism that probably results in a decrease in TSH. 71 .

[0137] Unlike DITPA, TRIAC increases D1 76 and further increases T3 when L-T4 is added. However, as a result, if serum T4, which functions as an essential precursor of T3 in the brain, decreases 71 , the effect of gene therapy may be weakened. Since all therapies have been used individually in the past, gene therapy combining both DITPA and TRIAC is expected to produce a combination of the effects observed with the individual therapies as described above. Furthermore, unbiased approaches such as RNAseq can be used to evaluate tissue transcriptional changes in the setting of combination therapy with AAV9-mediated gene therapy and TH analogs. 77

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[0139] The subject matter of this specification is not limited to the specific embodiments described above, but encompasses any embodiments within the scope of the general terms of the following claims enabled by the embodiments described herein, or any embodiments described in terms sufficient for one of ordinary skill in the art to make and use the claimed subject matter.

Claims

1. A pharmaceutical composition comprising a viral vector AAV9 for introducing normal human MCT8 into cells of a subject, for use in a method for treating Alan Herndon-Dudley syndrome in the subject, Method: To determine whether the subject requires treatment for Alan Herndon-Dudley syndrome, The method includes administering a composition to introduce normal human MCT8 into the target cells, thereby increasing T3 in the target brain and inducing a T3-mediated response, and performing gene therapy accordingly. A composition comprising normal human MCT8, a functional human MCT8 polypeptide encoded by the SLC16A2 gene, capable of transporting thyroid hormones across the cell membrane.

2. The composition according to claim 1, comprising a viral vector AAV9 for introducing normal human MCT8.

3. The composition according to claim 1, wherein the method further comprises administering DITPA to the subject.

4. The composition according to claim 3, wherein the administration of DITPA to the subject is initiated with a single dose over a period of two weeks and continued with a higher dose for at least two weeks.

5. The composition according to claim 3, wherein the single dose administered to the subject and the higher dose are determined based on the subject's T3 serum level.

6. The composition according to any one of claims 1 to 5, wherein the method further comprises administering TRIAC to the subject.

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