In an interview with OncLive, Mohamad Mohty, MD, PhD, discussed advancements in the management of graft-vs-host disease (GVHD) and highlighted unmet needs for patients with chronic (cGVHD) or acute GVHD (aGVHD), for whom standard-of-care therapies often fall short.

Mohty also expanded on ongoing research in GVHD reported at the 50th Annual EBMT Meeting. This included 3-year follow-up findings from the phase 2 ROCKstar trial (NCT03640481), which investigated belumosudil (Rezurock) in patients with cGVHD. The study showed that treatment with this agent yielded sustained responses, and no new safety signals were observed in eligible patients.1

Although challenges remain, advancements showcased at the EBMT meeting continue to underscore a new era for the treatment of patients with GVHD, Mohty noted. He provided further insights and key takeaways from the EBMT Annual Meeting in another interview with OncLive.

Mohty is a professor of hematology and the head of the Hematology and Cellular Therapy Department at Saint-Antoine Hospital and Sorbonne University in Paris, France. He is also head of the translational research team at the Saint-Antoine Research Center and the chairman of the Acute Leukemia Working Party of the EBMT.

Mohty: GVHD remains a matter of concern after transplant. Weve seen some major improvements over the past few years with the advent of posttransplant high-dose cyclophosphamide in particular. However, some patients are still [experiencing] aGVHD. Unfortunately, our first-line therapy remains focused on high-dose corticosteroids with deleterious adverse effects [AEs]; we also know that approximately 50% to 60% of patients are not going to respond to corticosteroids or are going to become corticosteroid dependent, which is not a good scenario.

Weve been lucky over the past 3 to 4 years to have the approval of the JAK2 inhibitor ruxolitinib, which proved to be a very good drug for patients who have steroid-refractory aGVHD. We are also struggling with a smaller group of patients who are becoming ruxolitinib refractory, resistant, or dependent. If you use ruxolitinib for salvage [therapy] after corticosteroids, a small group of patients may not be able to receive ruxolitinib, not everybody will respond, and some patients will lose their initial response to ruxolitinib. We then end up with a new group of patients who are ruxolitinib refractory. This is a new category of patients. Were fortunate that were now seeing research in this subgroup of patients. During the EBMT meeting, I was very excited to see some novel data on modulation of the microbiota in these patients with advanced and refractory aGVHD.

An important piece of information regarding the use of MaaT013 as salvage treatment based on the early-access program is its excellent safety profile. We didnt see severe AEs in the 140 patients who were treated. It is a single-shot treatment, which is important, instead of a treatment that is received and given to the patient on a chronic basis every day for several weeks or months.

Most importantly, the responses are very quickwithin 1 week. Patients who are responding are [doing so] in an excellent manner and very rapidly. Now based on this early-access program, we have very important follow-up that gives us some confidence in these results, which I consider quite reliable.

cGVHD incidence and severity have decreased over the past 10 years thanks to the efficacy of PTCy. However, we should bear in mind that were [performing transplant in] older patients. We are frequently using peripheral blood stem cells as well as donor lymphocyte infusion, including in the prophylaxis setting. At the end of the day, we still have a significant proportion of patients, at approximately 20%, who are continuing to experience moderate or severe cGVHD, which would require systemic immunosuppressive therapy.

Although cGVHD is not an immediate life-threatening condition, it dramatically alters the patients quality of life. Therefore, we still need to make progress in this arena. When it comes to the first-line therapy today, we dont have options better than corticosteroids. Thats the bad news because we dont like to use high-dose corticosteroids due to their deleterious AEs. We do have some good news about ruxolitinib because it was validated in the steroid-refractory cGVHD setting in the phase 3 REACH3 trial [NCT03112603].

But we have patients who may not be able to tolerate ruxolitinib; they may develop cytopenias over the long term or infections, or they may not respond to ruxolitinib. Therefore, novel options are needed, and this is where belumosudil, which was [granted regulatory] approved in 2021, is proving to be an excellent option. This drug has a new mechanism of action, as its a ROCK2 inhibitor. This mechanism of action is extremely interesting because when we look to the pathophysiology of cGVHD, we do have different phases. For instance, it is likely that ruxolitinib is acting well on the inflammatory component, which is an earlier phase, especially with the Th17 inflammatory response. But the use of a ROCK2 inhibitor such as belumosudil can also act not only on the inflammatory process but mainly on the fibrotic process. This is what makes it very attractive. This is what we saw in the ROCKstar trial, which allowed the approval of belumosudil in the United States and many other countries across the globe.

We can see more than a 70% response rate with belumosudil, and responses are in all organs usually involved with cGVHD, including the lung. [I stress] lung cGVHD because this is a terrible localization and [represents] a huge unmet need. Having a drug that [leads to] responses in the lung for cGVHD is most welcome. We are now getting closer to having better control of cGVHD. These drugs have been developed for the patient population with the most advanced, severe [disease]. The next step, and my wish, is to try to move these agents into earlier lines of use, even first-line treatment of cGVHD.

During the EBMT meeting we heard about the launch of a randomized trial with belumosudil in the first-line setting in combination with corticosteroids. The field is moving positively and, in my opinion, in the right direction.

Biomarkers in the field of GVHD, whether acute or chronic, are highly desired. I dont think anyone would question the need to have biomarkers to be able to predict the responses [and] also the failures, and maybe identify when to start treatment. Unfortunately, despite a lot of research and some very nice studies, Im not aware of 100% reliable, robust biomarkers in acute or chronic GVHD.

There have been some great work and results, for instance, with the Mount Sinai Acute GVHD International Consortium criteria and biomarker [research], but I still dont believe this is part of routine clinical practice. Its also true that in cGVHD, we dont have reliable biomarkers; maybe we will, but it will be difficult to find these biomarkers because cGVHD is a very heterogeneous condition. Clinically, cGVHD of the lungs or scleroderma may be different from dry eyes or dry mouth with cGVHD. In the ideal world, we would love to see these biomarkers. However, in the real world today, I dont see them ready for primetime yet.

Cutler C, Lee SL, Pavletic S, et al. Belumosudil for chronic graft-versus-host disease after 2 prior lines of systemic therapy: 3-year follow-up of the ROCKstar study. Presented at: 50th Annual Meeting of the EBMT; April 14-17, 2024; Glasgow, Scotland. Abstract OS13-01.

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Derivation of HOs from hiPSCs

We previously generated hiPSC-derived cardiac organoids (COs), and the CO formation resulted in the enhanced maturity of hiPSC-derived cardiomyocytes [18]. However, these COs could not mimic the diverse cellular composition of the human heart. Thus, we refined our differentiation protocol to generate heart organoids (HOs) designed to accommodate the coexistence of various cardiac lineage cells by modulating BMP, VEGF, FGF, and TGF signaling during differentiation (Fig. 1A).

A Overall schematic diagram of differentiation from hiPSC into COs and HOs. B Comparison of beating efficiency of COs and HOs at day 830 of differentiation. C The morphology of COs and HOs for 30 days, including the differentiation period.

An assessment of organoid beating efficiency spanned the entire differentiation period from day 8 to day 30 for both COs and HOs (Fig. 1B). On day 810 of differentiation, both COs and HOs initiated discernible beating (Fig. 1B). Notably, COs showed a beating efficiency of 68.1%, whereas HOs exhibited a lower rate of 34.5% on day 8 of differentiation. However, although the beating efficiency at the beginning of differentiation was higher in COs than in HOs, HOs displayed an ascending trend in their beating rate post-differentiation initiation, culminating in a 100% beating efficiency by day 30 (Fig. 1B), and resulted in similar beating efficiency to COs on day 30 (Fig. 1C, Videos 1 and 2).

Moreover, we validated the cardiomyocyte subtypes in the organoids by confirming the expression levels of MLC-2v and MLC-2a, indicative of ventricular and atrial types of cardiomyocytes, respectively. We observed that the expression of MLC-2a initiated early in differentiation (from D5 to D10), while MLC-2v expression gradually increased over the differentiation period. At D24, the majority of cardiomyocytes in both COs and HOs exhibited strong expression of MLC-2v (Fig. S1A and B).

To assess the cellular distribution in the COs and HOs, FACS analysis was performed (Figs. 2A, B and S2). Within COs, the mean distribution of cTnT, CD90, and VE-cadherin was 92.232.62%, 6.241.55%, and 4.731.48%, respectively (Figs. 2A, B and S2). Conversely, in HOs, these distributions were determined to be 51.095.92%, 24.634.08%, and 14.033.34%, respectively (Figs. 2A, B and S2).

A Representative pie chart showing the distribution of cTnT, a cardiomyocyte (CM)-specific marker, CD90, a cardiac fibroblast (CF)-specific marker, and VE-Cad, an endothelial cell (EC)-specific marker, in CO and HO. B The graph displays the mean cellular compositions of cardiomyocytes, fibroblasts, and endothelial cells across 11 different batches of COs and HOs. C Representative z-stack image of HOs and COs using confocal microscopy (left). Staining for cTnT (green) and VE-cad (red) in 2D monolayer culture of cells dissociated from HOs and COs (right). Scale bar: 100m. D Z-stack image of co-staining for Vimentin (fibroblast marker, green), -actinin (cardiomyocyte marker, red), and DAPI (blue) in COs and HOs. Scale bar: 100m. E Comparison of the gene expression levels for various cell types in COs and HOs. Quantitative analysis of gene expression levels as performed with real-time PCR. The expression levels of cardiomyocyte markers (NKX2.5, TNNT2, MYL2, and MYL7), endothelial cell markers (CD34, PECAM1, SOX17, and FOXA2), fibroblast markers (CD90, PDGFR, Vimentin, and TCF21) normalized to that of GAPDH. Data were shown as fold-change relative to COs, as meanSD, by 2-way ANOVA (n=3). A significant difference is indicated by #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001 compared with COs and ns (non-significant).

Immunostaining was also performed to visualize the distribution of cardiac lineage cells (Fig. 2C and D). Consistently with FACS analysis, COs prominently displayed cTnT expression within the organoids, while HOs displayed VE-cadherin expression on the surface of HOs (Fig. 2C, Videos 3, 4). The distinct distribution of blood vessel cells in HOs was further verified through the culture of dissociated organoids, and the dissociated cells from HOs exhibited a composition of both cTnT-positive cells and VE-Cad-positive cells (Fig. 2C). In contrast, the dissociated cells from COs exclusively displayed cTnT-positive cells (Fig. 2C). In addition, the HOs exhibited notable expression of Vimentin, a fibroblast marker, in comparison to COs (Fig. 2D).

The differences were further validated through qPCR analysis, and this analysis substantiated that COs exhibited a relatively increased expression of NKX 2.5, a transcription factor governing heart development, in addition to TNNT2, MYL2, and MYL7 (Fig. 2E). In contrast, HOs displayed significant upregulation in genes related to cardiac fibroblasts (CD90 and PDGFR), Vimentin, and TCF21, all in comparison to COs (Fig. 2E). Furthermore, endothelial-related genes including CD34, PECAM1, SOX17, and FOXA2 exhibited elevated expression levels in HOs relative to COs (Fig. 2E).

To gain a deeper understanding of the intricate gene expression within HOs, we utilized single-cell RNA sequencing (Fig. 3). Utilizing UMAP clustering and marker identification, the pool of 2587 cells from HOs was effectively categorized into distinct groups, encompassing cardiomyocytes, fibroblasts, and endothelial cells (Fig. 3A). To compare the representative gene expression patterns by cell type in both COs and HOs, we selected genes with p-values below 0.05 and visualized their expression using violin plots. Additionally, a comparative examination of gene expression patterns between COs and HOs was presented through violin plots. Notably, representative genes linked to cardiomyocytes, such as MYL2, MYL7, MYH7, TNNC1, MYBP3, and CACNA1C, exhibited enhanced expression in COs when compared to HOs (Fig. 3B). Similarly, an analysis was also extended to endothelial cells. Endothelial-related genes such as APOLD1, GIMAP4, PECAM1, PRSS23, STC1, and VEGFC exhibited an upregulation in HOs compared to COs (Fig. 3C). Shifting the focus to cardiac fibroblasts, the violin plots highlighted genes like AGT, CLU, and HMGA1 illustrating disparities between COs and HOs (Fig. 3D).

A Uniform manifold approximation and projection (UMAP) plots of HOs. Winseurat data sets labeled with Winseurat clusters. Detailed clustering within CMs (pink), CFs (green), and ECs (purple) clusters. BD Violin plots of representative genes for CMs (MYL2, MYL7, MYH7, TNNC1, MYBPC3, and CACNA1C), ECs (APOLD1, GIMAP4, PECAM1, PRSS23, STC1, and VEGFC), and CFs (AGT, CLU, and HMGA1). These genes were selected fold-change about 2-fold, average expression about 4 or more, and p-value 0.05 or less.

Clusters where unselected genes were grouped under the unclassified category in HOs were further analyzed (Fig. 3A). To predict cell types within the populations, we made use of databases such as pangiaoDB and GeneCard. These predictions revealed that the unclassified population mainly comprised pericytes, epithelial cells, neurons, and other cell types. These findings collectively indicate that HOs possess a more comprehensive genetic repertoire of heart constituent cells in comparison to COs.

With the established HOs, we mimicked the pathological conditions of acute myocardial infarction (AMI) and subsequent cardiac fibrosis to model human heart disease (Fig. 4A). To replicate hypoxia-induced ischemic conditions, we introduced 50M of cobalt chloride (CoCl2) [19] to the organoids along with glucose-depleted culture medium. This approach effectively elevated the expression of hypoxia-inducible factor-1 (HIF-1) in both COs and HOs (Figs. 4B and S3).

A Schematic of an experiment mimicking the heart disease in organoids by ischemia-reperfusion injury mechanism that occurs in the human adult heart and followed fibrogenesis. B Expression of the HIF-1 through western blots. Quantitative analysis of HIF-1 performed using Image J software. The expression of HIF-1 normalized to that of GAPDH. Data were shown as fold-change, and a significant difference is indicated by ****, ####p<0.0001, and ns (non-significant). C Immunofluorescence images of the expression levels of apoptotic marker (cleaved caspase-3, green) and cardiomyocyte marker (cTnT, red) in COs and HOs after IR injury. The scale bar represents 100m. D Representative western blot image and quantitative analysis of cleaved caspase-3. Data normalized to that of caspase-3. Equal protein loading amounts were confirmed by GAPDH expression. The corresponding density ratio was calculated by the average intensity of the bands from Image J software. Data were shown as fold-change, and a significant difference is indicated by ****, ####p<0.0001 and ns (non-significant). E Representative image and quantitative analysis of TUNEL assay (green). Data were shown as fold-change, as meanSD, by 2-way ANOVA (n=3). Significant difference is indicated by #p<0.05, ***p<0.001, ****, ####p<0.0001 (*compared to control group; #compared to COs), and ns (non-significant). The scale bar represents 100m.

In the clinical condition, the rapid reintroduction of blood flow post-reperfusion can lead to an immediate supply of oxygen and nutrients, triggering heightened inflammation and oxidative stress, thereby potentially causing tissue damage. A study suggested that high glucose sensitizes cardiomyocytes to ischemia-reperfusion (IR) injury [20]. Another study proposed that intracellular and mitochondrial calcium overload may contribute to reperfusion injury by exacerbating oxidative stress [21]. Based on these findings, we hypothesized that these factors could mimic reperfusion injury in CoCl2-treated HOs. To test this hypothesis, we applied a culture condition in which high glucose and calcium ion levels, then CoCl2-treated COs and HOs were exposed to a reperfusion medium rich in glucose and calcium ions for 72h.

To verify the induction of apoptosis in both COs and HOs following IR injury, we performed co-staining of cTnT and cleaved caspase-3 in organoid sections (Fig. 4C). This co-staining provided confirmation of the reduction in cardiomyocytes and the increase in apoptosis were more pronounced in HOs compared to COs (Fig. 4C). Western blot analysis against the cleaved caspase-3 further confirmed the more effective induction of apoptosis in HOs relative to COs (Figs. 4D and S4). Validation of apoptosis in the IR-injured organoids was also carried out using the TUNEL assay, and the findings demonstrated the more increased apoptotic cells within the HOs than that of COs (Fig. 4E). Additionally, Western blot analysis unveiled a more substantial increase in the Bax/Bcl2 signaling in HOs compared to COs (Fig. S5).

IR injury in humans leads to the disruption of sarcomere structures and a reduction in cardiac markers, including cTnT and cTnI, within heart tissue [22]. Furthermore, in clinical practices, markers such as cTnI, Myoglobin (MB), and Creatine kinase M (CKM) are quantified in blood to diagnose myocardial infarction resulting from IR injury [23].

Consistent with previous findings, we observed a more pronounced disintegration of sarcomere structures in HOs following IR injury, in contrast to COs (Fig. 5A). Simultaneously, the intracellular expression of cTnT and cTnI showed a marked reduction in HOs compared to COs (Figs. 5B and S6). Moreover, the release of cTnI, MB, and CKM from HOs began to be released from the organoids 24h post-IR injury, with the highest levels observed at 72h and exhibited significantly higher levels than those from COs (Fig. 5C).

A Immunofluorescence images of the expression levels of sarcomeric -actinin and DAPI in the control and IR groups. White dotted line in IR-induced HOs indicates the disintegrated sarcomere structure in the organoids. The scale bar represents 100m and magnified image scale bar represents 20m. B Western blot analysis in cell lysates from COs and HOs in control and IR groups. The protein expression of cTnT and cTnI, which are essential for cardiac structure was calculated by the average intensity of the bands from Image J software. The comparison of the fold change between the COs and HOs was normalized by the control group. C Extracellular secretion levels of cTnI, myoglobin (MB), and creatine kinase M type (CKM), which AMI indicators in control and IR group during culture periods. Secretion of cTnI, MB, and CKM was measured by ELISA. All data were shown as meanSD by 2-way ANOVA (n=3). Significant difference is indicated by ***, ###p<0.001, ****, ####p<0.0001(*compared to control group; #compared to COs), and ns (non-significant).

In addition, an analysis of inflammatory responses and necrosis-related mRNA levels in both COs and HOs following IR injury revealed a more notable increase in gene expressions within HOs in comparison to COs (Fig. S7A). Furthermore, HOs subjected to IR injury displayed a more significant upregulation of NF-B, a crucial transcription factor involved in inflammation and processes related to cardiac-vascular damage, in comparison to IR-injured COs (Figs. S7B and S8). The expression levels of phosphorylated ERK, phosphorylated JNK, and phosphorylated p38, which are indicative of increased signaling pathways in cardiac remodeling post-AMI [24, 25], were also significantly elevated in IR-injured HOs compared to COs (Figs. S7C and S8).

Intracellular calcium overload and subsequent mitochondrial calcium accumulation are observed in acute myocardial ischemia, and these phenomena are exacerbated during reperfusion, ultimately leading to mitochondrial permeability transition pore (mPTP) opening [26]. Furthermore, within a physiological environment, the bulk of calcium during cycles of contraction and relaxation is released from and taken up by the sarcoplasmic reticulum (SR) [27]. Therefore, quantifying SR calcium content is essential in elucidating the pathophysiological mechanisms of calcium overload.

We first observed calcium overload in the organoids by measuring the activity of sarco/endoplasmic reticulum calcium ATPase (SERCA), which significantly influences SR calcium storage [28], to infer the SR calcium content. Before IR injury, there were no significant differences in basal and peak intracellular calcium concentrations between COs and HOs during the contractionrelaxation cycle (Fig. 6A and B). However, after IR injury, both the basal and peak calcium concentrations were significantly higher in HOs compared to COs (Fig. 6A and B). Subsequently, we measured the activity of SERCA (kSERCA) by calculating the time constant after inhibiting SERCA and observed that the SERCA activity was notably increased in IR-injured HOs compared to IR-injured COs (Fig. 6C). In the same context, the phosphorylated phospholamban (PLN) expression was significantly elevated in IR-injured HOs, confirming the accelerated SERCA activity (Fig. 6D and S9). This suggests that SERCA activity preferentially increases during IR induction in HOs, leading to a significant increase in SR calcium storage in IR-injured HOs compared to IR-injured COs.

A Representative trace of calcium transient in COs and HOs before and after IR injury. control and IR groups. B Basal and peak Ca2+ concentrations were measured using calcium imaging. All data were shown as meanSD by 2-way ANOVA (n=1820). A significant difference in all graphs are indicated by *, #p<0.05, **, ##p<0.01, ***, ###p<0.001, ****, ####p<0.0001 (*compared to control group; #compared to COs), and ns (non-significant). C The SERCA rate constant, reflecting the activity of SERCA, was calculated by subtracting the reciprocal of the time constant measured after inhibiting SERCA from the reciprocal of the time constant measured in the transient. All data were shown as meanSD by 2-way ANOVA (n=1820). A significant difference in all graphs are indicated by *, #p<0.05, **, ##p<0.01, ***, ###p<0.001, ****, ####p<0.0001 (*compared to control group; #compared to COs), and ns (non-significant). D Western blot analysis of phospholamban and phosphorylated phospholamban in COs and HOs before and after IR injury. Quantitative analysis of all western blot data was calculated by the average intensity of the bands in Image J software. Equal protein loading amounts of western blot data were confirmed by GAPDH expression. A significant difference of all graphs is indicated by #,*p<0.05, ##,**p<0.01 ###,***p<0.001, ####,****p<0.0001(*Compared to control group; #compared to COs), and ns (non-significant). E Representative immunofluorescence images of MPTP opening (calcein, green) in control and IR groups. The scale bar represents 200m. F MPTP opening (calcein) ratio in each group was calculated by image J software. This data was normalized to the control of COs. G Beating characteristics of COs and HOs in IR and control groups. Beating analysis was performed by monitoring calcium fluorescence over a period of 20s under control and IR conditions. A comparison of BPM (beat per minute), peak-to-peak duration, and time-to-peak was performed on COs and HOs in each group. All data were shown as meanSD by 2-way ANOVA (n=3). A significant difference in all graphs is indicated by **, ##p<0.01, ***, ###p<0.001, ****, ####p<0.0001 (*compared to the control group; #compared to COs), and ns (non-significant). H Schematic summary of findings in (AF).

To determine whether the IR condition facilitates mPTP opening in HOs, we directly measured fluorescence intensity using an mPTP assay kit in the IR-injured organoids and found a more significant reduction in fluorescence intensity within IR-injured HOs compared to COs (Fig. 6E and F), suggesting that IR injury in HOs leads to a greater increase in mPTP opening than IR injury in COs.

Moreover, the real-time calcium transient assay allowed for an analysis of calcium handling properties in IR-injured organoids (Figs. 6G and S10). Amplitudes showed no significant difference between COs and HOs during ischemia and IR injury (Fig. 6G). However, HOs subjected to ischemia-injury exhibited aberrant beating properties (Fig. 6G) with a significant change in beating rate (peak-to-peak) and systolic time (time-to-peak), indicated a disease-like model. Consistent with the ischemia results, the IR-injury condition induced further detrimental calcium handling properties in HOs, resulting in a decrease in beating and systolic parameters compared to those of COs (Fig. 6G). Collectively, these results indicate that multicellular HOs effectively mimic clinically observed AMI pathologies, such as calcium overload and mPTP opening under IR conditions, as well as the mimicking defects in calcium handling function (Fig. 6H).

Cardiac fibrosis is a consequential outcome of cardiac remodeling following AMI [29]. To replicate the cardiac fibrosis within IR-injured HOs, we cultured the organoids with 10M TGF-1 for 7 days. Staining for COL1A1, a marker indicating fibrosis progression, revealed a more pronounced accumulation of collagen in IR-injured HOs after fibrosis induction compared to IR-injured COs (Fig. 7A and S12A). This substantial collagen accumulation in IR-injured HOs was further confirmed through western blot analysis and Massons trichrome (MT) staining (Fig. 7B, C and S12B). Additionally, we assessed increased expression levels of mRNA associated with fibrosis-related genes (ACTA2, POSTN, Vimentin, MMP2) and collagen-related genes (PAI1, COL1A1, COL1A2, COL3A1) in IR-injured HOs after fibrosis induction using quantitative PCR (Fig. S12C).

A Representative immunofluorescence images of COL1A1 (green) and DAPI (blue) in each group. The scale bar represents 100m. B The protein expression of COL1A1 and -SMA, which are fibroblast activation and fibrosis indicators using western blot in cell lysates from COs and HOs in each group. Equal protein loading amounts were confirmed by GAPDH expression. C The morphologies of the IR-Fibrosis organoid by Massons Trichrome staining. The scale bar represents 40m. D Evaluation of the electrophysiological function of COs and HOs on the electrode of the MEA plate in each group. Magnified image to show a heatmap of a representative MEA recording. The spike activity of each active electrode is color-coded: white/red represents high spike activity; blue/black represents low spike activity. E Beating rate (BPM), Spike amplitude, FPDcF, and conduction velocity of COs and HOs in each group through MEA recording. All data were shown as fold-change, as meanSD, by 2-way ANOVA (n=3). A significant difference of all graphs is indicated by #,*p<0.05, ##,**p<0.01, ###,***p<0.001, ####,****p<0.0001(*Compared to control group; #compared to COs) and ns (non-significant). F Comparison of contraction in control COs versus IR-fibrosis COs and control HOs versus IR-fibrosis HOs.

To validate the alterations in calcium handling in HOs under the IR-fibrosis condition, we conducted a calcium transient assay. Real-time video recordings allowed for an analysis of beating properties (Fig. S13A and Videos 11, 12) of the organoids, and HOs subjected to IR-fibrosis displayed an increase in amplitude and beating (peak-to-peak) but a decrease in systolic (time-to-peak) compared to IR-fibrosis COs (Fig. S13B), reflecting a form of arrhythmic event in the human heart.

Heart disease also leads to alterations in electrophysiological properties [30, 31]. To demonstrate the defect of electrophysiological characteristics in IR-fibrosis HOs, multielectrode arrays (MEA) were utilized (Fig. 7D). Consistent with the analysis of beating properties in calcium transient, the beating rate (BPM) was significantly increased in IR-fibrosis HOs compared to IR-fibrosis COs, and the BPM exceeded 100, a characteristic of tachycardia (Fig. 7E). The spike amplitude, indicative of action potential height, demonstrated a decline in both COs and HOs within the IR-fibrosis group, but HOs displayed a particularly significant difference compared to COs (Fig. 7E). Measurement of the field potential duration corrected by Fridericias formula (FPDcF) unveiled a twofold increase in HOs subjected to IR-fibrosis (Fig. 7E), mirroring the prolongation of the period between the onset of the Q wave and the conclusion of the T wave. The cardiac conduction velocity exhibited a significant slowing in IR-fibrosis HOs compared to IR-fibrosis COs, resulting from an elevated risk of re-entrant excitation attributing to collagen accumulation in HOs (Fig. 7E). Additionally, induction of fibrosis subsequent to IR injury resulted in reduced contractility of COs but maintained normal cardiac rhythms, whereas, in HOs, it led to both diminished contractility and irregular cardiac rhythms (Fig. 7F).

The QuantSeq 3 mRNA-Sequencing analysis provided further support for the modeling of AMI and cardiac fibrosis in HOs (Fig. 8). In comparing the up-regulated KEGG pathways between control HOs and IR-injured HOs (Fig. 8A), it was observed that the FoxO signaling pathway, known to be activated in cardiomyocytes under ischemic stress [32], was predominantly up-regulated. Additionally, pathways associated with cancer, which share common systemic pathology and mechanisms with heart failure [33], were also up-regulated in IR-injured HOs. Furthermore, pathways related to extracellular matrix (ECM)-receptor interaction, PI3K-Akt signaling, and HIF-1 signaling were up-regulated in IR-injured HOs compared to control HOs which are known to play crucial roles in cardiac remodeling [34], alleviating negative post-infarct changes in myocardium [35], and modulating post-infarct healing after myocardial ischemic injury [36], respectively. Interestingly, pathways associated with human papillomavirus, insulin resistance, efferocytosis, longevity genes, and focal adhesion kinase (FAK) inhibition were also found to be up-regulated in IR-injured HOs. These pathways are implicated in various processes such as the diagnosis of myocardial infarction [37], hypoxia-induced inhibition of angiogenesis [38], macrophage-mediated clearance of dead cells during myocardial infarction [39], modulation of cardiovascular function [40], and regulation of cardiac fibrosis post-MI [41].

A Visualized graphs of up-regulated KEGG pathways in IR-injured HOs compared to control HOs. B Visualized graphs of up-regulated KEGG pathways in IR-fibrosis HOs compared to control HOs. The pathways were selected based on criteria including a fold change >2 and a p-value less than 0.05.

The comparison between control HOs and IR-fibrosis HOs revealed up-regulated pathways associated with cardiac fibrosis (Fig. 8B). One of the pathways identified as up-regulated in IR-fibrosis HOs, is the calcium signaling pathway, known to play a role in fibroblast activation by increasing intracellular calcium concentration, promoting fibroblast proliferation and migration, and inducing the synthesis of extracellular matrix proteins [42]. Extracellular matrix (ECM) remodeling is closely linked to cardiac remodeling and the development of heart failure [43]. Furthermore, the renin-angiotensin-aldosterone system (RAAS) pathway, Wnt signaling pathway, and adrenergic signaling pathway were also identified as up-regulated in IR-fibrosis HOs. These pathways are known to promote myocardial fibrosis and cardiac remodeling, contributing to the progression of heart failure [44,45,46,47], respectively. Additionally, conditions such as Cushing syndrome, circadian disruption, and cardiomyopathy, which are associated with increased myocardial fibrosis [48,49,50], were found to be relevant to the up-regulated pathways in IR-fibrosis HOs. Cortisol is also experimentally shown to induce cardiomyocyte hypertrophy [51].

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Injections of cardiac spheroids into primate ventricles improved left ventricular ejection after four weeks.

Researchers from Shinshu University and Keio University School of Medicine have tested a novel strategy for regenerative heart therapy. They transplanted cardiac spheroids derived from human induced pluripotent stem cells (HiPSCs) into damaged ventricles and observed very positive outcomes in primate models. These results could expand treatment options for people suffering from heart problems.

The prevalence of myocardial infarction is rising. These destroy millions of cardiac muscle cells, leaving the heart in a weakened state. Currently, as mammals cannot regenerate cardiac muscle cells on their own, heart transplants are the only clinically viable option for patients suffering heart failure. However, full heart transplants are expensive and donors are rare, so alternative therapies are highly sought after.

The team cultivated HiPSCs in a medium that led to their differentiation into cardiomyocytes. Following the extraction and purification of cardiac spheroids, they injected approximately 6 107cells into the damaged hearts of crab-eating macaques and monitored the condition of the animals for twelve weeks, taking regular measurements of cardiac function.

Analysis of the monkeys hearts at the tissue level was then conducted to assess whether cardiac spheroids could regenerate the damaged heart muscles. The researchers verified the correct reprogramming of HiPSCs into cardiomyocytes first, observing at cellular-level electrical measurements that the cultured cells showed patterns typical of ventricular cells. Also, the cells responded as expected to numerous known drugs. Significantly, they discovered that the cells abundantly expressed adhesive proteins like connexin 43 and N-cadherin, which would promote their vascular integration into an existing heart.

Furthermore, this approach is less expensive and easier to adopt because the cells were transported from the production facility at Keio University to Shinshu University, located 230km away. The cardiac spheroids were preserved at 4C in standard containers and withstood the four-hour journey, meaning extreme cryogenic measures would not be required when transporting the cells to clinics.

The monkeys received injections of either cardiac spheroids or a placebo directly into the damaged heart ventricle. The team noted that arrhythmias were very uncommon, with only two individuals experiencing transient tachycardia in the first two weeks among the treatment group. Echocardiography and computed tomography exams confirmed that, compared to the control group, the hearts of monkeys that received treatment had better left ventricular ejection after four weeks, demonstrating a superior blood pumping capability.

Ultimately, it was revealed through the histological analysis that the cardiac grafts were mature and properly connected to pre-existing existing tissue, confirming the results of previous observations. HiPSC-derived cardiac spheroids could potentially serve as an optimal form of cardiomyocyte products for heart regeneration, given their straightforward generation process and effectiveness, explained first author Dr Hideki Kobayashi. We believe that the results of this research will help solve the major issue of ventricular arrhythmia that occurs after cell transplantation and will greatly accelerate the realisation of cardiac regenerative therapy.

Despite this cardiac spheroid production protocol being tested in monkeys, it was designed for clinical application in humans. The favourable results obtained thus far are sufficient to provide a green light for our clinical trial, called the LAPiS trial. We are already employing the same cardiac spheroids on patients with ischemic cardiomyopathy, concluded Dr Kobayashi.

This study was published in Circulation.

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Accelerating cardiac regenerative therapy with HiPSC spheroids - Drug Target Review

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In December 2014, Dr. Stephen Coles, a UCLA professor who studied aging, passed away from pancreatic cancer. While for many years Coles had made his home in Los Angeles, he chose to enter hospice care in Scottsdale, Arizona. That way, he could be close to the team of doctors who would

Once Coles was pronounced dead, that team arrived at his bedside. They restored his breathing and blood circulation with a heart-lung resuscitator, also known as a thumpera mechanical device used in emergency medicine to perform CPRand injected his body with anticoagulants to keep the blood flowing. All of this was done to protect the brain from damage that can occur after too long without oxygen. Next, the body was cooled in an ice water bath, the blood replaced with an organ preservation solution.

Finally, Coles body arrived at its final destination: Alcor, the nations oldest provider of cryonics, the freezing of human corpses and brains in liquid nitrogen that will one daytechnology willinglive again.

There, surgeons performed a neuroseparation, removing Coles head at the sixth cervical vertebra, and pumped cryoprotectants (medical-grade anti-freeze) into the now severed head. Then, a forensic pathologist opened the skull and removed the brain.

Coles had died around 10 a.m.; by dinner time, his brain was in a silver dewar, its thermostat set to -140 degrees Celsius.

The Patient Care Bay at Alcor holds a number of Bigfoot dewars, which are custom-designed to contain four whole-body patients and five neuropatients each. The dewar is an insulated container which consumes no electric power. Liquid nitrogen is added periodically to replace the small amount that evaporates.

Coles was Alcors 131st patient but one of its first to select brain-only cryopreservation, sometimes called neuropreservation or neurosuspension. A company announcement called Cole an unusual brain-only patient, and revealed that the unfamiliar nature of the procedure created several major challenges, with procedures being revised even as the surgery and perfusion were underway.

Ten years later, according to Emil Kendziorra, M.D., CEO of Tomorrow Bio, a German biotech firm that specializes in human cryopreservation, brain removal is not a big issue, and becoming more popular among those interested in cryonics. Storing a brain is faster, cheaper, anddespite the human taboo of decapitationpoised for a higher degree of social acceptance, Dr. Kendiziorra says, since anatomy departments and research institutions have been storing brains for years.

But what about the rest of the body? Wont future humans need their legs and arms when they wake up from their cryonic suspension?

While the brain is unique and cannot be recreated, the fundamental logic is that all the rest of the body can be recreated, Dr. Kendziorra tells Popular Mechanics. This means that by the time technology exists to cure death and reanimate the human brain, slapping together a real or virtual vessel should be a cinch.

These ideas may seem far-fetched, but Dr. Kendziorra is quick to point out that there was a time in the past when heart transplantationtaking one heart and connecting into another bodysounded pretty science fiction as well.

But as neuropreservation grows in popularity, the question remains: what will we do with all of these frozen brains in the future?

***

Dr. Kendziorra is a trained medical doctor-turned-cryonics evangelist. As a former cancer researcher, he was frustrated by the agonizingly slow pace of progress and never found it acceptable to tell a 25-year-old that they have incurable cancer and theyre going to die, he says. I think that everybody should live as long as they choose to.

Its important to point out that no human brain (or whole human for that matter) has ever been revived after death. The hope behind cryonics is that, eventually, very smart people using technology that hasnt been invented yet will figure out how to conquer death. For anyone with an untreatable diseaseor anyone who would like to live beyond their average lifespanto elongate their lives, they just need to freeze themselves, and then wait for those smart (and hopefully benevolent) people to wake us up.

Its also important to point out that freeze is the wrong word. Technically, cryonically preserved bodies arent frozen, theyre vitrified. Youve probably heard that the human body is 70 percent water; if you popped a corpse in the freezer, there would be a lot of cracking when ice crystals formed in the cells and damaged the body beyond revival. Upon thawing, the body would be mushy, thanks to the ruptured cell walls caused by cracking.

The Alcor operating theater in Scottsdale, Arizona. Here, surgeons perform initial procedures to gain access to the patients vascular system, replacing the blood with a cryoprotectant solution to prevent the formation of ice crystals during subsequent cooling.

Instead, cryopreservation involves vitrification, replacing the blood with a medical antifreeze, called cryoprotectant, then cooling the body gradually until it resembles glass.

The cost of these proceduresas well as transporting the body and storing it for untold yearsis not cheap. For full-body suspension, Tomorrow Bio charges 200,000. Thats why, although he always recommends full-body cryopreservation, Dr. Kendziorra says that, at the bargain price of 75,000, brain-only cryopreservation is an attractive option to those hoping to extend their time on Earth.

Dr. Kendziorra says he feels strongly about making cryopreservation possible at more price points, but its not just humans hoping to live forever that will benefit from increasing affordability. The field of cryonics needs an infusion of cash and research funding if it is going to maintain long-term storage facilities and figure out how to cure death. Theoretically, more bodiesor brainsin more tanks will lead to a greater investment in these endeavors by the scientific community.

Today, Alcors membership is split nearly evenly between whole-body and neuro cryopreservation. While neurosuspension is easier and less expensive, there are still some compelling reasons to consider whole-body cryopreservation.

For one thing, no one can be sure that the brain contains everything we would need to feel like ourselves upon reanimation. Without the central nervous system, the spine, the endocrine glands, and microbiome, would we recognize ourselves upon waking in the distant future?

Its a concern that led Becca Ziegler, a 23-year-old Tomorrow Bio member, to opt for whole-body preservation. From my understanding, everything that makes me me is in the brain, she says, but there are still some unknowns about consciousness and memories and how the brain interacts with the rest of the body. So I chose whole-body cryopreservation to ensure that there are no essential parts of my consciousness and memories that arent cryopreserved.

Dr. Kendziorra says that out of an abundance of caution, his company always recommends whole-body cryopreservation, unless its not within the budget of a perspective member. After all, rousing from a cryonic state hundreds of years in the future with only half of your identity would be a real disappointment. Better safe than sorry, says Dr. Kendziorra.

***

According to Dr. Kendziorra, there are currently four working theories for what future generations will do with thawed-out human brains. All of this is very speculative, he warns, but they have potential.

The first and perhaps most realistic, based on existing technology, is 3D printing.

We could 3D print all the other organs and connect the brain, Dr. Kendziorra says. This technology isnt there quite yet, but its probably not that far away anymore. Indeed, 3D organ bioprintingthe use of human cells to create three-dimensional tissueis a quickly evolving field, fueled by the hundreds of thousands of people who need organ transplants. Jennifer Lewis, a professor at Harvard Universitys Wyss Institute for Biologically Inspired Engineering, predicts the technology could be ready in a decade.

Another possibility will be the development of clones using DNA taken from brain tissue. The clone, of course, will need to be created without a brain, so that the old one can be transplanted. Since the birth of Dolly the sheep in 1996, scientists have cloned 22 animal species as well as a human embryo. Could brainless vessels be next?

Some scientists believe clones wont be necessary, and that reanimated brains could be transplanted in donor bodies, a method neurosurgeon Sergio Canavero called technically feasible, in a recent paper (published in a journal he is an editor of, it should be mentioned). After detailing how the cranial nerve and vascular system could theoretically be reconnected to the brain, the controversial scientist admitted there was lots of work still ahead, including cadaveric rehearsals, tests in brain-dead organ donors, and the development of new surgical tools. With appropriate funding, he argued, a long-held dream may finally come true.

The third way a reanimated brain could once again express itself is by being placed in an artificial body. In simpler terms, a robot body, says Dr. Kendziorra. Elon Musk thinks its possible and so does Michael S.A. Graziano, a Princeton neuroscientist. Graziano argued in a Wall Street Journal essay that uploading a mind into a robot body would take only two pieces of technology: an artificial brain and a scanning device with the ability to measure exactly how [a brains] neurons are connected to each other, to be able to copy that pattern in the artificial brain.

Then again, the robot might not even be necessary. We could reinstantiate the brain by connecting it to a computer, and all sensation inputs and outputs would be virtual, Dr. Kendziorra explains. On some abstract level, maybe theres not much a difference between real and virtual. Hes got a point; some scientists already believe were living in a simulation.

Regardless of just what future humans do with cryonically preserved brains, Dr. Kendziorra believes its going to take a long time to figure it out. Medically and technologically we are not there yet, and we will not be there for many, many decades. Its going to take a significant amount of time. And in fact, it might never work.

But if theres a reason to stay hopeful about cryonics, Dr. Kendziorra says its because the other option isnt so great either. The alternative, he laments, is death.

Ashley Stimpson is a freelance journalist who writes most often about science, conservation, and the outdoors. Her work has appeared in the Guardian, WIRED, Nat Geo, Atlas Obscura, and elsewhere. She lives in Columbia, Maryland, with her partner, their greyhound, and a very bad cat.

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The Science of Frozen Heads: How the First Cryonic Brains Will Rise Again - Popular Mechanics

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Cell culture

Human MSCs from bone marrow were purchased from Riken BioResource Research Center (Ibaraki, Japan)and cultured in Dulbeccos Modified Eagles Medium (DMEM: Sigma-Aldrich, St. Louis, MO, USA) with 10% fetal bovine serum (FBS, Sigma-Aldrich). Cells were passaged four to five times before use for transplantation. HK-2 cells, a human proximal tubular cell line, were obtained from the American Type Culture Collection (Manassas, VA). These cells were cultured as described previously47.

MSCs were pretreated with or without recombinant human IFN- (PeproTech, Cranbury, NJ, USA) by the following method. When MSCs reached 70% confluence, IFN- was added to the medium to achieve a final concentration of 10ng/mL. After 48h, cells were collected and subjected to in vivo and in vitro analyses.

Male Sprague Dawley (SD) rats (8weeks old) were purchased from Charles River Laboratories Japan (Yokohama, Japan). Experimental procedures were approved by the Institutional Animal Care and Use Committee of Hiroshima University (Hiroshima, Japan) (Permit Nos. A15-66 and A17-75) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals, 8th ed, 2010 (National Institutes of Health, Bethesda, MD, USA). This study is reported in accordance with ARRIVE guidelines. To establish the animal model, SD rats were randomly divided in 6 groups (n=5 in each group): sham, PBS (control), MSCs, IFN- MSCs, NC siRNA/IFN- MSCs and IDO1 siRNA/IFN- MSCs groups. All procedures were performed under anesthesia with injection of agents composed of midazolam, medetomidine, and butorphanol. Right nephrectomy was performed 7days prior to IRI of the left kidney. Renal IRI was induced by transiently clamping the unilateral renal artery. After a laparotomy was performed, the left kidney was exposed. Next, the renal pedicle was clamped by atraumatic vascular clamps for 45min, followed by reperfusion on a heating blanket. After reperfusion, phosphate-buffered saline (PBS, vehicle), control MSCs, or IFN- MSCs (5105 cells/rat) were injected through the abdominal aorta clamped above and below the left renal artery bifurcation. At 7 or 21days post-injection, rats were sacrificed and their kidneys were collected to evaluate inflammation and fibrosis.

Immunohistochemical staining was performed according to previously described methods47 using the following primary antibodies: mouse monoclonal anti-Foxp3 (Abcam, Cambridge, UK), rabbit polyclonal anti-CD3 (Dako, Glostrup, Denmark), mouse monoclonal anti-rat CD68 (Serotec, Oxford, UK), and rabbit polyclonal anti-collagen type I (Abcam). FOXP3-, CD3- and CD68-positive cells, as well as areas positive for -SMA and collagen type I staining, were assessed using ImageJ software (version 1.53s, NIH) by examining five randomly selected fields (100magnification) of the cortex.

Double immunostaining was performed according to the following methods. Sections of formalin-fixed, paraffin-embedded tissues (4m thick) were de-paraffinized, subjected to heat-mediated antigen retrieval in citric acid buffer at 98C for 40min, and then blocked in 5% skim milk at room temperature for 1h. They were incubated with anti-FOXP3 antibody (Abcam) overnight at 4C, followed by incubation with the appropriate secondary antibody (DAKO) at room temperature for 1h, and then incubated with 3,3-diaminobenzidine (Sigma-Aldrich) at room temperature for 5min. After that, they were heated again in EDTA buffer (pH 9.0) in the same way. They were then blocked in 2.5% normal horse serum (ImmPRESS Horse Anti-Rabbit IgG Polymer kit; Vector Laboratories, Riverside, CA, USA) at room temperature for 20min, followed by incubation with anti-CD3 antibody (Abcam) overnight at 4C. They were incubated with the secondary antibody (ImmPRESS Horse Anti-Rabbit IgG Polymer kit; Vector Laboratories) at room temperature for 30min and then incubated with working solution prepared with Vector SG Peroxidase (HRP) Substrate Kit (Vector Laboratories) at room temperature for 5min.

Sections of formalin-fixed, paraffin-embedded tissues (2m thick) were stained with Massons trichrome to assess fibrosis. Areas of interstitial fibrosis were assessed using Lumina Vision (Mitani, Osaka, Japan) by examining five randomly selected fields (100magnification) of the cortex.

Sample collection and western blotting were performed as previously reported36,47 with the following primary antibodies: anti-VEGFA antibody (Abcam), mouse monoclonal anti--SMA (Sigma-Aldrich), rabbit monoclonal anti-TGF-1 (Abcam), IDO1 polyclonal antibody (Proteintech, Rosemont, IL, USA), mouse monoclonal anti-Foxp3 (Abcam), rabbit polyclonal anti-CD4 (Abcam), and mouse monoclonal anti-GAPDH (Sigma-Aldrich). Horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Dako) or goat anti-mouse immunoglobulin G (Dako) were used as secondary antibodies. SuperSignal West Dura or Pico Systems (Thermo Fisher Scientific, Waltham, MA, USA) were used to detect signals. The intensity of each band was analyzed by ImageJ software and standardized to the level of GAPDH.

To generate conditioned medium (CM) from untreated MSCs (control MSCs-CM) and IFN- MSCs (IFN- MSCs-CM), human MSCs (3105 cells/dish) were seeded in 10-cm dishes and cultured in DMEM containing 10% FBS. When the cells reached at least 70% confluence, the medium was replaced with fresh medium with or without 200ng/mL recombinant human IFN- (PeproTech). After 48h, the culture medium was replaced with DMEM containing 0.1% FBS, which was collected after 48h.

RNA extraction and real-time reverse-transcription PCR were conducted according to previously described methods47. Specific primers and probes for human IDO1 (assay ID: Hs00984148_m1), and human -actin (assay ID: Hs99999903_m1) were obtained as TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA). mRNA levels were normalized to the level of -actin.

ELISA analysis of IDO (R&D Systems, Minneapolis, MN, USA) was performed according to the manufacturers protocol. Concentrations were normalized to the total protein content.

Human peripheral blood mononuclear cells (PBMCs; Biosciences, Berkeley, CA, USA) were suspended with the buffer formulated as MACS BSA Stock Solution (Miltenyi, Bergisch Gladbach, NRW, Germany) and autoMACS Rinsing Solution (Miltenyi). Cells were labelled with a Nave CD4+T Cell Isolation Kit II (Miltenyi) according to the manufacturers protocols. Nave CD4-positive T cells were sorted by negative selection using LS columns (Miltenyi) and MidiMACS (Miltenyi), and then collected.

Nave CD4 T cells (1106 cells/mL) were cultured in RPMI-1640 (Solarbio, Beijing, China) plus 0.1% FBS (Thermo Fisher Scientific) with MSCs-CM or IFN- MSCs-CM at a RPMI-1640:CM ratio of 1:1. Next, Dynabeads human T cell activator CD3/CD28 (Thermo Fisher Scientific) was added at a bead:cell ratio of 1:1, along with animal-free human recombinant IL-2 (ProteinTech) at a concentration of 300IU/mL, and cells were incubated in a humidified CO2 incubator. The medium, IL-2, and beads were exchanged on day 3, and then cells were collected on day 5.

MSCs were transfected with 20nM siRNA against IDO1 (s7426, Applied Biosystems) or negative control siRNA (4390843, Applied Biosystems) using Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific). After 24h, transfected cells were washed and fresh complete medium was added. When cells reached 80% confluence, they were collected and subject to in vivo experiments.

Results are expressed as the meanstandard deviations (S.D.). For multiple group comparisons, one-way ANOVA followed by Bonferronis post-hoc test was applied. Comparisons between two groups were analyzed by Students t-test. P<0.05 was considered statistically significant.

All experimental procedures were approved by the Institutional Animal Care and Use Committee of Hiroshima University (Permit Nos. A15-66 and A17-75).

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Mesenchymal stem cells pretreated with interferon-gamma attenuate renal fibrosis by enhancing regulatory T cell ... - Nature.com

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A placebo-controlled Phase 1/2 trial conducted in East Shanghai has found that administering umbilical cord-derived mesenchymal stem cells reduces frailty in older people.

These researchers begin by defining frailty as a state of heightened vulnerability to potential stressors as a consequence of reduction in physiological reserves across multiple systems [1]. This vulnerability destroys the strength and endurance of older people, exhausting their stamina and greatly increasing their risks of death and disability, and a metric has been determined to measure this [2]. However, while vitamin supplements may help people with nutritional deficiencies, there are no medically approved drugs to treat frailty [3].

As stem cell exhaustion has been pinpointed as a cause of frailty [4], replacement stem cells have been investigated as a possible treatment. In particular, mesenchymal stem cells (MSCs), which are naturally attracted to injury sites [5], appear to be the most promising. MSCs have multiple potential sources for derivation [6], and previous trials have been conducted to treat frailty by using MSCs derived from bone marrow (BM-MSCs), with positive results [7, 8].

This study, however, was conducted on stem cells that were originally derived from the human umbilical cord (HUC-MSCs). These cells are easy to mass produce [9], have been successfully clinically tested against other diseases such as heart failure [10] and arthritis [11], and fight inflammation [12]. This, however, is the first trial of HUC-MSCs for frailty.

All participants had to meet three criteria: to be between the ages of 60 and 80, to score between 1 and 4 on the Fried frailty scale [2], and to be expected to live another year. A large variety of co-morbidities were screened out, such as uncontrolled diabetes, serious cardiovascular problems, infections, and viral diseases. This was a double-blinded trial from which 80 potential candidates were excluded. 15 patients received placebo, and 15 received MSCs, for 6 months.

This study measured physical performance by testing grip strength, the timed up-and-go test, walking speed, and the ability to stand up and sit back down. Inflammatory cytokines such as interleukins were also measured, and sleep quality, quality of life, and mental health were also assessed.

There were no significant adverse effects. Three participants had suffered from ailments during the trial, two of which were in the placebo group and the third of which had dizziness not related to the MSCs.

Physical function, the primary endpoint of the study, was strongly affected by the MSCs. Even with only 30 total participants, not all of which participated in every assessment, the researchers were able to obtain, against baseline, a p-value of .003 after only one week of treatment and p-values under .001 for 1 and 6 months. Against placebo, the p-value at the end of the 6-month study was .042.

There were possible effects on mental health and sleep quality but those could be statistically attributed to the placebo effect. However, the treatment improved total quality of life with a p-value of 0.002 against placebo at the end of the study.

Cytokines had less clear effects; the placebo group spiked in TNF- and IL-17 at 6 months while the MSC group did not.

While not all of the endpoints were hit, this study was against frailty, and it is clear from these results that MSCs have beneficial impacts on frailty in human beings. However, this study was conducted in one country among 30 people. Further work, with a larger sample size and more testing sites, will need to be conducted to determine if these results hold up under further scrutiny.

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[1] Clegg, A., Young, J., Iliffe, S., Rikkert, M. O., & Rockwood, K. (2013). Frailty in elderly people. The lancet, 381(9868), 752-762.

[2] Fried, L. P., Tangen, C. M., Walston, J., Newman, A. B., Hirsch, C., Gottdiener, J., & McBurnie, M. A. (2001). Frailty in older adults: evidence for a phenotype. The Journals of Gerontology Series A: Biological Sciences and Medical Sciences, 56(3), M146-M157.

[3] Dent, E., Morley, J. E., Cruz-Jentoft, A. J., Woodhouse, L., Rodrguez-Maas, L., Fried, L. P., & Vellas, B. (2019). Physical frailty: ICFSR international clinical practice guidelines for identification and management. The Journal of nutrition, health and aging, 23(9), 771-787.

[4] Schulman, I. H., Balkan, W., & Hare, J. M. (2018). Mesenchymal stem cell therapy for aging frailty. Frontiers in Nutrition, 5, 108.

[5] Golpanian, S., Wolf, A., Hatzistergos, K. E., & Hare, J. M. (2016). Rebuilding the damaged heart: mesenchymal stem cells, cell-based therapy, and engineered heart tissue. Physiological reviews, 96(3), 1127-1168.

[6] Zhang, J., Huang, X., Wang, H., Liu, X., Zhang, T., Wang, Y., & Hu, D. (2015). The challenges and promises of allogeneic mesenchymal stem cells for use as a cell-based therapy. Stem cell research & therapy, 6, 1-7.

[7] Golpanian, S., DiFede, D. L., Khan, A., Schulman, I. H., Landin, A. M., Tompkins, B. A., & Hare, J. M. (2017). Allogeneic human mesenchymal stem cell infusions for aging frailty. Journals of Gerontology Series A: Biomedical Sciences and Medical Sciences, 72(11), 1505-1512.

[8] Tompkins, B. A., DiFede, D. L., Khan, A., Landin, A. M., Schulman, I. H., Pujol, M. V., & Hare, J. M. (2017). Allogeneic mesenchymal stem cells ameliorate aging frailty: a phase II randomized, double-blind, placebo-controlled clinical trial. Journals of Gerontology Series A: Biomedical Sciences and Medical Sciences, 72(11), 1513-1522.

[9] Sarugaser, R., Lickorish, D., Baksh, D., Hosseini, M. M., & Davies, J. E. (2005). Human umbilical cord perivascular (HUCPV) cells: a source of mesenchymal progenitors. Stem cells, 23(2), 220-229.

[10] Bartolucci, J., Verdugo, F. J., Gonzlez, P. L., Larrea, R. E., Abarzua, E., Goset, C., & Khoury, M. (2017). Safety and efficacy of the intravenous infusion of umbilical cord mesenchymal stem cells in patients with heart failure: a phase 1/2 randomized controlled trial (RIMECARD trial [randomized clinical trial of intravenous infusion umbilical cord mesenchymal stem cells on cardiopathy]). Circulation research, 121(10), 1192-1204.

[11] Wang, L., Huang, S., Li, S., Li, M., Shi, J., Bai, W., & Liu, Y. (2019). Efficacy and safety of umbilical cord mesenchymal stem cell therapy for rheumatoid arthritis patients: a prospective phase I/II study. Drug design, development and therapy, 4331-4340.

[12] Uccelli, A., Pistoia, V., & Moretta, L. (2007). Mesenchymal stem cells: a new strategy for immunosuppression?. Trends in immunology, 28(5), 219-226.

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Stem Cell Treatment Restores Strength in Trial - Lifespan.io News

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27-year-old Kerri Paton was overjoyed to welcome her first child with husband Igor Topas, 28, a little over six years ago. Baby Amelia was born a healthy 6lbs 12oz. But shortly after Kerri gave birth, her doctor noticed the newborn had pale purple spots all over her body.

Once she was in my arms I just cried. I was so happy, Kerri recalls of the birth to UK news site yahoo!life.

Acute means that these types can progress rapidly.

RELATED: 11-Month-Old Baby Boy of Beloved Female Police Officer Is Diagnosed with AML Leukemia as Community Rallies in Support Fighting Pediatric Cancer

Amelia had to begin chemotherapy at just three weeks old. Luckily, after the first round of chemotherapy, Kerri saw that her babys lumps and spots were gone, though she still had a long way to go.

Amelia also endured a bone marrow transplant, or stem cell transplant, which is a procedure where healthy cells are transplanted into your blood or bone barrow.

After eight months of treatment, she was declared in remission by her medical team.

Six months later after hearing the joyous news of remission, during a routine follow-up, Kerri and Igor tragically learned that their baby girl had relapsed with AML. Igor had been on his way to the appointment when the doctor called Kerri and said she should come in as well.

I just knew it had come back, Kerry said.

What Are The Symptoms of Relapse in Acute Myeloid Leukemia (AML)?

In addition to more chemotherapy, Amelia needed another stem cell transplant.

Thankfully, the transplant worked, and Amelia has now been cancer free for five years. Just before Amelias recurrence, the Scottish couple had another baby, a son named Oscar, so theyre now a happy and healthy family of four.

Watching your kid sick it was horrible, Kerri said of all they have endured. At six years old, she noted that her daughter is just so funny and fiercely independent. Shes very strong-willed.

Added Kerri, Even though she went through cancer, her story doesnt end there.

Acute myeloid leukemia (AML) is a cancer that affects bone marrow, the spongy tissue inside of your bones. Its a rare cancer overall, but it is the most common type of leukemia in adults. Children rarely get AML.

Dr. Mikkael Sekeres, Director of the Cleveland Clinic Cancer Center Leukemia Program, Explains How AML Works

This disease is caused by DNA damage to the cells in your bone marrow that give rise to blood cells. Red blood cells carry oxygen to tissues, white blood cells fight infections as part of the immune system, and platelets help stop bleeding. Those cells are damaged in AML, and the damage results in an overproduction of unnecessary white blood cells.

Acute lymphoblastic leukemia (ALL) is a type of leukemia where the bone marrow makes too many immature lymphocytes, a type of white blood cell. It is also called acute lymphocytic leukemia, according to the National Cancer Institute.

Dr. Olalekan Oluwole, a hematologist with Vanderbilt University Medical Center, previously spoke with SurvivorNet about ALLs effect on the body and the type of treatments that work to fight it.

ALL is a type of cancer that is very aggressive, Dr. Oluwole told SurvivorNet. It grows very fast. Within a few weeks, a few months, the person will start to feel very sick. And thats why we will have to give it an equally aggressive type of treatment to break that cycle.

All About Acute Lymphoblastic Leukemia

Dr. Oluwole also says the leukemia often resides in the bone marrow, and because it is an abnormal growth, it just keeps dividing.

It doesnt follow rules, and it doesnt stop, he told SurvivorNet. Not only that, because this is part of the immune system, the immune system is sorta like the police of the body. So those abnormal cells that have now become cancer, they have the ability to go to many places. They go into the blood, and they often go into the tissue or the lining around the brain.

As parents navigate their young ones cancer journey, its important to remember that childrens bodies may react differently to treatments because their bodies are still growing.

They may receive more intense treatmentsand they may respond differently to drugs that control symptoms in adults, the National Cancer Institute informs. Be sure to ask a lot of questions.

RELATED: Why Do Pediatric Drugs Take So Long to Develop? A Look into the Lag Time on Drug Approvals for Childhood Cancer & Other Illnesses

Remember, youre not alone your childs oncologist and care team are there to guide you and provide information and answers. Oncological social workers can also be a vital resource to help you sort out the financial aspects of cancer treatment, as well as other cancer-related issues. Skilled psychologists and counselors can be accessed to help you maintain good mental health through your childs cancer journey, to the best of your ability.

Additionally, dont be afraid to reach out to your support system friends, relatives, etc. for help through this process. No one expects you to handle everything on your own.

Meanwhile, if youre wondering what you can do to ensure your child is getting the best treatment possible, consider the following recommendations from theNational Cancer Institute.

At SurvivorNet, we always encourage people to advocate for themselves when it comes to cancer and, more generally, healthcare. When it comes to a child, the parent must become the advocate.

RELATED: The Top Ten Childhood Cancer Symptoms That Can Be Missed

Its important to speak up about each and every issue that may concern you, no matter how minor, as even minor signs can sometimes clue doctors in on a potential cancer diagnosis. And catching it as early as possible is always ideal, as early detection may help with treatment and outcomes.

When It Comes to Health, Its Okay to Be a Little Pushy

Seeking multiple opinions is one way to make sure you or your child is getting the proper care and attention. You should also try to remember that not all doctors are in agreement. Recommendations for further testing or treatment options can vary, and sometimes its essential to talk with multiple medical professionals.

Every appointment you leave as a patient, there should be a plan for what the doc is going to do for you, and if that doesnt work, what the next plan is, Dr. Zuri Murell, director of the Cedars-Sinai Colorectal Cancer Center, previously told SurvivorNet. And I think that thats totally fair. And me as a health professional thats what I do for all of my patients.

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According to Market.us, the Mesenchymal Stem Cells Market Size is expected to achieve a value of around USD 10 billion by the year 2033. This indicates a noteworthy escalation from its 2023 valuation of USD 3 billion. Such substantial growth is forecasted to occur at a Compound Annual Growth Rate (CAGR) of 12.6% during the projection period spanning from 2024 to 2033.

The Mesenchymal Stem Cells Market is undergoing significant transformations, influenced heavily by its interconnectedness with various end-use industries. These industries are pivotal in shaping the Mesenchymal Stem Cells Markets dynamics, as they drive demand and set stringent quality standards. The alignment between the market offerings and the industries evolving needs ensures a consistent demand, fostering a scenario ripe for sustained growth in the Mesenchymal Stem Cells sector. This interdependence necessitates that market players remain agile, innovative, and responsive to the shifting requirements and emerging trends within these pivotal sectors.

Regulatory frameworks set by governments worldwide are integral to the Mesenchymal Stem Cells Markets structure, influencing its operational, environmental, and compliance standards. These regulations ensure the markets adherence to safety, quality, and sustainability norms, which are increasingly becoming stringent. The adherence to these standards in the Mesenchymal Stem Cells Market is not just about legal compliance but also about building trust with consumers and maintaining a competitive edge. The markets resilience is thus tied to its ability to navigate the complex regulatory landscape, adapt to new laws, and uphold the highest standards of operational excellence.

The Mesenchymal Stem Cells Markets dynamics are further shaped by the intricate import-export mechanisms and the flow of investments. Changes in trade policies, import-export regulations, and international tariffs directly influence the Mesenchymal Stem Cells Markets stability and growth trajectories. Investment from both governmental and private sectors plays a critical role, underpinning innovation and technological advancements in the Mesenchymal Stem Cells arena. These investments, along with strategic initiatives like mergers, acquisitions, and partnerships, are pivotal in driving the market forward, enabling scalability, and enhancing its global outreach.

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In this market research, Market.us uncovered key insights that offer actionable takeaways and provide a clear direction for future market strategies. Mesenchymal Stem Cells market findings reveal critical trends and developments that shape the market landscape. These insights equip businesses with valuable information to make informed decisions and stay ahead of the competition. By understanding consumer preferences, market dynamics, and emerging opportunities, companies can optimize their product offerings, refine their marketing strategies, and capitalize on growth prospects. Mesenchymal Stem Cells research highlights the importance of staying agile and adaptable in response to evolving market conditions. With these key takeaways, businesses can confidently navigate the market landscape, mitigate risks, and drive sustainable growth in the long term.

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In market research, its essential to identify and understand various market segments to tailor strategies effectively. By delineating the key market segments within the Mesenchymal Stem Cells market, businesses can refine their approach to cater to specific customer groups. This segmentation allows for more targeted marketing efforts, product development, and customer relationship management. Through thorough analysis, industries can identify common characteristics, needs, preferences, and behaviors within each segment. Mesenchymal Stem Cells insights enable companies to craft tailored messaging, promotions, and offerings that resonate with the unique needs of each segment. Moreover, understanding Mesenchymal Stem Cells market segments facilitates resource allocation, helping businesses allocate their resources efficiently and maximize their return on investment. Overall, identifying and targeting key market segments is crucial for businesses seeking to effectively engage with their target audience and achieve sustainable growth.

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When researching the Mesenchymal Stem Cells industry, its crucial to understand and leverage factors that drive growth. These may include technological advancements, increasing consumer demand, and supportive government policies. By recognizing and capitalizing on these forces, Mesenchymal Stem Cells industry can position themselves strategically to capitalize on growth opportunities. However, its also important to address market restraints such as regulatory challenges, economic downturns, and shifting consumer preferences. By identifying these obstacles early on, businesses can develop strategies to mitigate their impact and navigate through challenges effectively.

Additionally, exploring untapped Mesenchymal Stem Cells market opportunities and emerging trends is essential. This involves identifying new market segments or niche markets and developing targeted strategies to capture these opportunities. Staying informed about Mesenchymal Stem Cells market trends, including shifts in consumer behavior, technological innovations, and the competitive landscape, is crucial for maintaining a competitive edge. Overall, comprehensive market research involves analyzing internal and external factors to make informed decisions and drive sustainable growth within the Mesenchymal Stem Cells industry.

The regional analysis of the Mesenchymal Stem Cells market provides valuable insights into its performance across various geographical areas, offering a comprehensive understanding of the opportunities and challenges present in each region. By examining factors such as economic conditions, regulatory frameworks, consumer preferences, and competitive landscapes, researchers can identify key trends and dynamics shaping Mesenchymal Stem Cells market dynamics at the regional level. This analysis enables stakeholders to tailor their strategies and investments to capitalize on specific market nuances and maximize growth potential. Moreover, understanding regional variations allows companies to mitigate risks associated with Mesenchymal Stem Cells market fluctuations and adapt their approaches to effectively target diverse customer segments. Overall, a robust regional analysis serves as a vital tool for informed decision-making and successful market penetration strategies.

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In assessing the competitive landscape of the Mesenchymal Stem Cells market, it is essential to analyze key players strengths, weaknesses, and strategies. Leading companies in the healthcare sector typically have robust distribution networks, strong brand recognition, and diversified product portfolios, which are their primary strengths. However, they may also face challenges such as fluctuating market demand, regulatory constraints, and competitive pricing pressures. Strategies employed by Mesenchymal Stem Cells industry players often include product innovation, strategic partnerships, mergers and acquisitions, and market expansion initiatives. By continuously leveraging their strengths and addressing weaknesses, these companies strive to maintain or enhance their market position while adapting to evolving industry dynamics. A comprehensive understanding of the competitive landscape enables stakeholders to make informed decisions and develop effective strategies to capitalize on Mesenchymal Stem Cells market opportunities.

Recent developments in the Mesenchymal Stem Cells market, including mergers, acquisitions, and product launches, are shaping the industry landscape. These events reflect strategic maneuvers by companies to gain competitive advantage and expand their market presence. Mergers and acquisitions often lead to market consolidation and portfolio diversification, while new product launches drive innovation and address evolving consumer demands. Staying informed about Mesenchymal Stem Cells developments is crucial for understanding market dynamics and identifying opportunities for growth and investment.

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Health & Wellness

May 2, 2024

Hormone therapy is a treatment that uses medications containing hormones like estrogen or progesterone to replace those the body stops producing during menopause. It can be a powerful tool for managing menopausal symptoms.

"While hormone therapy can significantly improve quality of life and overall health for many women, its not a one-size-fits-all solution," says Dr. Taryn Smith, a Mayo Clinic internist and womens health specialist.

She emphasizes the importance of a personalized medicine approach to hormone therapy, allowing healthcare providers to consider your health and potential risks.

Having discussions around menopause is important because it's often not a topic that is openly discussed or acknowledged, despite its significance and importance, she says. Menopause is a natural biological process, marking the time that a woman ends her menstrual cycle. It's diagnosed after a woman goes 12 months without a menstrual period.

"Many women dont know how to navigate menopause and the symptoms that come along with it, such as hot flashes and night sweats. Hormone therapy is used to manage many of these symptoms," says Dr. Smith. "But it's not for everybody."

Watch: Dr. Taryn Smith talks about hormone replacement therapy

Journalists: Broadcast-quality sound bites with Dr. Smith are available in the downloads at the bottom of the posts. Name super/CG: Taryn Smith, M.D./Internal Medicine/Mayo Clinic

Menopause symptoms and hormone therapy Hormone therapy is used to manage symptoms of menopause, such as hot flashes, night sweats, trouble sleeping, and irritability.

"For the average healthy woman, these options are very safe. But if there are any concerns about these risk factors, its best to consult your physician or your womens health specialist," she says.

Treatment options Hormone therapy is a prescribed medication that can come in different forms, including patches, gels, sprays and oral medications.

Those options may include "A patch you wear on the skin, almost like a bandage in the lower groin or over the buttocks. We have gels that you can rub into the inner thigh and sprays that can be sprayed on the forearm. We also have some oral options. With all the options available, typically, we can find a good option that meets the average woman's needs," says Dr. Smith.

Expected outcomes After starting hormone therapy, women can expect relief from symptoms. In many cases, hot flashes can be completely resolved, improving the womans quality of life.

"Often, we're able to completely resolve hot flashes. But if we cannot completely resolve them, we hope to get significant relief at least and help a woman restore her quality of life," she says.

Safety concerns While there are safety concerns associated with hormone therapy, such as potential risks of breast cancer, stroke, or heart disease, it is generally safe for the average woman who is close to the menopause transition and younger than 60.

"For the average healthy woman, these options are very safe. But if there are any concerns about these risk factors, it's best to consult your physician or your women's health specialist," says Dr Smith.

For those who are unable to take hormone therapy or choose not to, there are FDA-approved nonhormonal treatments for menopause symptoms. Talk with your healthcare team to find out what is the best option.

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Hormone therapy: Four things a Mayo Clinic women's health specialist wants you to know - Mayo Clinic

Recommendation and review posted by Bethany Smith

Hormone therapys benefits to treat menopausal symptoms outweigh the risks for women under 60. And its safer than previously thought, according to a new study published in JAMA, the journal of the American Medical Association. But the use of menopausal hormone therapy is not recommended to prevent heart disease, stroke, dementia or other chronic illnesses. It doesnt reduce those risks.

The study says roughly 55 million women in the U.S. and 1.1 billion worldwide are postmenopausal. A national coalition of researchers launched the Womens Health Initiative, which is the biggest study of womens health in the country, enrolling 161,808 postmenopausal women ages 50 to 79, to inform clinical practices around aspects of health impacting older women. Recruitment for the study began in 1993 and each participant was followed for up to 20 years.

For years, it was believed that hormone replacement therapy lowered the risk of heart disease, stroke, dementia, chronic disease and even death. But as United Press International reported, one of the Womens Health Initiatives clinical trials slammed the brakes on hormone replacement therapy in 2002, however, reporting that women taking combination (estrogen and progestin) hormone therapy had an increased risk for breast cancer, heart disease, stroke and blood clots.

But they continued to study the issue, as the popularity of hormone replacement therapy dropped and many doctors stopped prescribing it. The article said that a more nuanced picture of hormone therapys risks and benefits has emerged as the research continued.

The article said that research over time has continued to show that hormone therapy doesnt solve age-related risks like heart disease or hip replacements, which other early studies suggested. But its not as risky as believed for younger women bothered by symptoms as they approach menopause. And in menopause, hormone replacement therapy can offer relief from those symptoms. .

Per NPR, Most significantly, there are now different types of hormones delivered at lower doses that are shown to be safer.

Women should know that hormone therapy is safe and beneficial, Dr. Lauren Streicher, a clinical professor of obstetrics and gynecology at Northwestern University Feinberg School of Medicine, told NPR.

The same studys findings do not support routinely recommending women take calcium plus vitamin D supplements to prevent fractures, but these supplements are appropriate to fill nutritional gaps for women who dont get enough of those nutrients through their diet, the researchers said.

They also noted that a low-fat dietary pattern with increased intake of fruits, vegetables and grains did not prevent breast or colorectal cancer, but was associated with lower rates of death from breast cancer in long-term follow-up, offering an option for women seeking to reduce this risk, Dr. JoAnn Manson, chief of Preventive Medicine at Brigham and Womens Hospital and first author of the new report in JAMA, said in a news release. Women also have more options for treatment now, including estrogen in lower doses and delivered through the skin as a patch or gel, which may further reduce risks; non-hormonal treatments are also available.

The Mayo Clinic News Network talked with Dr. Taryn Smith, one of Mayos internists and womens health specialists, about the value of a personalized approach to medicine when it comes to hormone replacement therapy. Smith offered four things women should know:

More here:
For women under 60, no reason to fear hormone replacement therapy - Deseret News

Recommendation and review posted by Bethany Smith

Back in 2003, preliminary

But the findings of the study were later debunked, and other researchers discovered that the original study looked at women who were 65 and up who already had a greater risk of heart attack, stroke, blood clots, and more, which ended up skewing the data. The study also didnt factor in how old the women were when they started hormone therapy, which further muddled the results.

Now, a longitudinal follow-up to the Womens Health Intitiative (WHI) study is out in JAMAand the findings suggest that women in menopause are just fine to take hormone therapy.

The WHI findings should never be used as a reason to deny hormone therapy to women in early menopause with bothersome menopausal symptoms, lead study author JoAnn E. Manson, M.D., chief of the division of preventive medicine at Brigham and Womens Hospital and the Michael and Lee Bell Professor of Womens Health at Harvard Medical School, said in a press release. Many women are good candidates for treatment and, in shared decision making with their clinicians, should be able to receive appropriate and personalized healthcare for their needs.

Dr. Mason also noted that women have more options for treatment now, including receiving estrogen in lower doses and delivered through the skin as a patch or gel which may further reduce risks.

But the study findings stop short of saying that hormone therapy will help with health issues associated with menopause, like bone loss, dementia, and heart disease, although other studies have found it may be helpful. Thats caused several doctors who treat women in menopause to be frustrated. Heres what you need to know.

The new study looked at long-term follow-up data of up to 20 years from the Womens Health Initiative. It found that women below the age of 60 had lower rates of adverse events and a more favorable benefit-to-risk ratio of hormone therapy than women in later menopause. Meaning, they got more out of doing hormone therapy than not taking it.

The study results found that women in early menopause had less moderate-to-severe hot flashes, night sweats, and other symptoms of menopause when they were on hormone therapy. However, they concluded that hormone therapy should not be used to prevent heart disease, stroke, dementia, or other chronic diseases.

The study also looked at calcium and vitamin D supplements, concluding that these should not be recommended to prevent fractures in all postmenopausal women. However, researchers also found that calcium and vitamin D supplements may be helpful to fill in nutritional gaps for women who dont get enough in their diet.

The researchers also concluded that a low-fat diet with more fruits, vegetables, and grains didnt reduce the risk of being diagnosed with breast or colorectal cancer, but it was linked with a lower risk of dying from breast cancer.

Hormone therapy and hormone replacement therapy are terms that are usually used interchangeably. Both terms are used to describe a medical treatment that may help relieve the symptoms of menopause and perimenopause (the period before menopause), according to the American College of Obstetricians and Gynecologists (ACOG). This can also be referred to as menopausal hormone therapy.

The Menopause Society uses the term hormone therapy to acknowledge that were not trying to replace pre-menopause levels of hormones, says Lauren Streicher, M.D., a clinical professor of obstetrics and gynecology at Northwestern University Feinberg School of Medicine. But hormone replacement therapy is a term that most women are familiar with. Doctors will often use the term menopausal hormone therapy when discussing this topic, though, Dr. Streicher says.

You could make an argument that hormone therapy applies to treatments like estrogen vaginal cream for vaginal dryness that has no systemic benefits, but theyre usually used interchangeably, says G. Thomas Ruiz, M.D., lead ob/gyn at MemorialCare Orange Coast Medical Center in Fountain Valley, CA.

There are mixed emotions about this study. This is really not astonishingly new information, says Mary Jane Minkin, M.D., a clinical professor of obstetrics and gynecology and reproductive sciences at Yale School of Medicine and founder of Madame Ovary. We know from previous studies that hormone therapy is quite safe in women proximate to menopause and that indeed it is the most effective intervention for women suffering from many menopausal symptoms.

But, despite its benefits, many women are scared of the idea of taking hormone therapy given the findings of the original Womens Health Initiative study. This study can help with reassuring women that hormone replacement therapy is, in fact, beneficial, says womens health expert Jessica Shepherd, M.D., an ob/gyn in Texas and author of the upcoming book on menopause, Generation M. However, there needs to be a more demonstrative approach to show the benefits and how to use hormone replacement therapy preventatively.

But some doctors are frustrated with these findings. Saying that this study should never be used as a reason to deny hormone therapy? Guess what? Thats exactly what the effects of the original study were, Dr. Ruiz says.

Dr. Streicher calls the study infuriating, adding, theyre taking data that was poorly designed and flawed in the first place and are commenting on it.

Dr. Streicher also says the conclusion that hormone therapy shouldnt be used to lower the risk of heart disease and breast cancer is tricky, given that other studies have found hormone therapy can be a helpful tool for lowering risk. Dr. Minkin agrees.

The one major anxiety of American women is breast cancer, she says. Even in the initial WHI studies, the group of women who were taking estrogen alone never showed any increased risk of breast cancer on estrogen alone; indeed they showed a reduced risk of breast cancer. And followup long-term studies on estrogen plus progestin showed no increases in mortality from estrogen and progestin. So, we have very good documentation of safety.

For prevention, we do know that estrogen therapy is protective against developing osteoporosis, Dr. Minkin says. We also know that for very young women, particularly those under the age of 45 who were not studied in the WHI studythat looked exclusively at women over 50that estrogen therapy is vital to help protect those very young women against heart disease and dementia, she says. But that data is not addressed in this particular study because the Womens Health Initiative study only looked at women over the age of 50, Dr. Minkin says.

What to do if youre interested in hormone therapy for menopause

There is still a lot of misinformation tied to the original Womens Health Initiative study, and some doctors are not up to speed on current data surrounding the benefits of hormone therapy, Dr. Ruiz says.

Thats why he and other ob/gyns recommend seeing a specialist if youre interested in taking hormone therapy for menopause. See someone who knows what they are talking about and understands the data, Dr. Streicher says. They should be able to help you find the right treatment based on your personal circumstances and goals.

Its up to patients and their healthcare providers to determine if HRT is right for them. Dr. Manson tells Prevention that the follow-up WHI study was designed to provide clarification to healthcare providers on the use of hormone therapy in menopausal women. Ive heard from women across the country saying that theyre having trouble finding someone who will still prescribe hormone therapy, she says. Were saying very clearly from the WHI investigators who know the trials extremely well that these findings should not be used as a reason to deny hormone therapy to women seeking treatment of bothersome menopausal symptoms.

Korin Miller is a freelance writer specializing in general wellness, sexual health and relationships, and lifestyle trends, with work appearing in Mens Health, Womens Health, Self, Glamour, and more. She has a masters degree from American University, lives by the beach, and hopes to own a teacup pig and taco truck one day.

Read more:
Study: Hormone Replacement Therapy Helps Menopause Symptoms - Prevention Magazine

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Nothing will have you Googling HRT therapy faster than your first hot flash.Hormone Replacement Therapy, or HRT, can be a lifesaver for many women navigating the challenges of menopause. It helps ease those sneaky hot flashes, stabilizes wild mood swings, and can even mitigate some of the physical changes that come with the change. Yet with all the options available, deciding on the right path can feel daunting. While HRT offers various treatment methods, each comes with its own set of risks and benefits.

One of the biggest concerns surrounding HRT is the potential for side effects and health risks. These can range from minor irritations to more severe complications, depending on the delivery method and individual health factors. The risks of taking hormone therapy differ for each individual, says Cincinnati, OH OB/GYN Somi Javaid, MD. Factors like the type, dose, duration of use, route of administration, and timing of initiation all play a role in determining safety and effectiveness.

Hormone therapy can be delivered systemically, affecting the entire body, or locally, targeting specific areas. According to Dr. Javaid, systemic therapy is usually aimed at addressing broader menopausal symptoms, while local therapy targets specific issues like vaginal dryness. Systemic therapy can be delivered orally or via transdermal patches, gels, sprays, or pellets, she explains.

Oral tablets are the simplest method, but Dr. Javaid warns about higher risks like blood clots and vascular events associated with this form of delivery. Transdermal patches, on the other hand, provide a consistent hormone release and are generally considered safer due to a reduced risk of clotting issues, she notes.

Hormone gels and sprays are another option you can apply directly to the skin. These allow for more precise dosing says Dr. Javaid, but cautions, They require careful application to avoid unintentional transference to others.

Implants or pellets, which are inserted under the skin, offer a steady hormone release over several months. However, Duxbury, MA plastic surgeon Christine DiEdwardo, MD points out that Dosage adjustments with pellets can be challenging since once a pellet is placed, it cannot be easily removed.

Each HRT delivery method has its pluses and minuses.Willowbrook, IL dermatologistJessie Cheung, MDnotes the convenience and flexibility of some: The advantage of oral and topical therapies is that you can quickly adjust the dosing, as most are taken daily and patches are applied every few days. However, this flexibility can come at a cost. The disadvantage with pellets is that most pellet procedures are done every three to six months. So dosing cant be adjusted easily. There also some soreness for a few days after, Dr. Cheung explains.

Pellets can be convenient due to their long-lasting nature, but they do require a minor surgical procedure for insertion. For many patients, the convenience of pellets outweighs the inconvenience of a 5-minute, in-office procedure, says Dr. Cheung.

However, Lauren Streicher, MD,clinical professorofobstetricsandgynecologyatNorthwestern Universitys Feinberg SchoolofMedicine says the cons of pellets are many. Pellets are not FDA approved and not recommended by most academic menopause experts, she explains. Every single professional society, including ACOG and The Menopause society has a very clear statement saying that pellets are unregulated, result in dangerously high levels of hormones and are associated with bleeding, uterine pre-cancer, cancer increased rates of hysterectomy and other side effects.

Conversely, creams offer quick adjustments in dosage but can be messy and require daily application. Creams can transfer to clothing or be passed to others and theres variable absorption, adds Dr. DiEdwardo.

The pros of transdermalestrogenin the form of patches, creams, gels and sprays: since they are not metabolized by the liver and unlike oral estrogen, do not increase the risk of blood clots or gallbladder disease, notes Dr. Streicher.Dr. Javaid adds that one thing to consider with patches is they might not be ideal for individuals who have adverse reactions to adhesives or those who dont want others to know theyre using HRT.

The best form of HRT is one that the patient is comfortable with and compliant in using, Dr. Cheung says. The choice is influenced by several factors, including health history, lifestyle and specific symptoms. Dr. Javaid advises considering your health history and any medications youre currently taking. The risks of taking hormone therapy differ for each individual, depending on type, dose, duration of use, route of administration and timing of initiation, she adds.

Patients should also consider their daily routines and personal preferences. Dr. Cheung mentions that some patients grow weary of applying creams twice a day, while others find pellet insertion procedures uncomfortable.

Discuss your options with your healthcare provider to determine which method aligns best with your needs and lifestyle, says Dr. Javaid.

There have been shown to be benefits of HRT, however there are also potential risks and contraindications to hormone therapy. Therefore factors that are evaluated prior to HRT include underlying medial history and risk of certain diseases, like cancer, heart disease, stroke, and blood clots, age at the start of hormone therapy, type and route of administration, dosage and how long you are on the medication, notes Dr. DiEdwardo. A thorough medical and family history, discussion of symptoms, physical exam and extensive testing are part of the evaluation prior to initiating HRT.

Read this article:
Pellets, Patches, Sprays and Creams: All the Ways to Get Hormone Replacement Therapy - NewBeauty Magazine

Recommendation and review posted by Bethany Smith

AI-enabled protein design company Profluent has leveraged artificial intelligence to design an open-source gene editor called OpenCRISPR-1, demonstrating the technology can be used to create molecules with the power to edit human DNA.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology, developed more than a decade ago, allows scientists to modify DNA sequences within living organisms precisely.

Potential applications range from treatments for genetic disorders to researching disease mechanisms.

The molecules it designs are fully synthetic and do not exist in nature, in contrast to previous technologies in gene editing, such as CRISPR-Cas9.

The company is open-sourcing OpenCRISPR-1 for free ethical research and commercial use and published the science behind the protein's development in a preprint publication.

"Attempting to edit human DNA with an AI-designed biological system was a scientific moonshot, Ali Madani, Profluent cofounder and CEO, said in a statement. "Our success points to a future where AI precisely designs what is needed to create a range of bespoke cures for disease."

WHY THIS MATTERS

AI was at the heart of this achievement, with the company training large language models (LLMs) on massive scale sequence and biological context.

The Profluent team developed a database of 5.1 million Cas9-like proteins, and the AI model was trained on this database to create potential proteins for CRISPR use.

This enabled the LLM to create novel gene editors from scratch as it learned through examples found in nature.

After narrowing down the results, they identified OpenCRISPR-1, a protein performing similarly to Cas9 but with far less impact on off-target sites. This makes it more precise and causes minimal damage to DNA.

The goal of open-sourcing OpenCRISPR-1 is to encourage the use of AI for ethical research and commercial use, particularly in developing medicines leveraging CRISPR.

"We believe by doing so, we can help accelerate the pace of discovery and innovation in the field," Madani said. "Our vision is to move biology from being constrained by what can be achieved in nature to being able to use AI to design new medicines precisely according to our needs."

He added that the company intends to partner with cutting-edge research institutions and drug developers working across the drug development lifecycle to enable CRISPR medicines to become available to a greater number of patients and for a greater number of disorders.

THE LARGER TREND

Gene editing technologies, including SHERLOCK and DETECTR, are transforming digital diagnostics, enabling rapid detection of infectious diseases such as COVID-19.

Companies including Atomwise, Deep Genomics and Valo are incorporating gene editing into drug discovery processes, revolutionizing treatment development.

Beyond gene editing, AI is powering everything from bone marrow analysis software to drug discovery and platforms to help pair patients with the right cancer-treatment drugs.

Originally posted here:
Profluent releases AI-enabled OpenCRISPR-1 to edit the human genome - Mobihealth News

Recommendation and review posted by Bethany Smith

Paralog meta-analysis

To reanalyze the data from the 5 paralog screens, raw read counts were downloaded, and the same pipeline was applied to all of them. A pseudocount of 5 reads was added to each construct in each replicate, and total read counts were normalized to 500 reads per construct. Log2 fold change (LFC) for each guide at late time point was calculated relative to the plasmid sequence counts.

The data from each study (except Thompson) were divided into three groups; the constructs that target single genes paired with non-essential/non-targeting gRNAs (N) in the first position (gene_N), in the second position (N_gene) and constructs that target gene pairs (A_B). LFC values of each group were scaled individually so that the mode of each group was set to zero. Next, all three groups were merged in one table. Before dividing Itos dataset into three groups, LFC values were scaled such that the mode of negative controls (non-essential_AAVS1) would be zero and also TRIM family was removed from this dataset to avoid false paralog pair discovery13. Since in Thompsons study there was just one position for singleton constructs, LFC values were scaled so that the mode of negative controls (non-essential_Fluc) was set to zero. In the next step, LFC of each construct was calculated by the mean of LFC across different replicates.

To calculate genetic interaction, single gene mutant fitness (SMF) was calculated as the mean construct log fold change of gene-control constructs for each gene. The control was either non-essential genes or non-targeting gRNAs. For each gene pair, the expected double mutant fitness (DMF) of genes 1 and 2 was calculated as the sum of SMF of gene 1 and SMF of gene 2. The difference between expected and observed DMF, the mean LFC of all constructs targeting genes 1 and 2, was called dLFC.

Next step was calculating a modified Cohens D between observed and expected distribution of LFC of gRNAs targeting genes. Expected distribution of gRNAs targeting a gene pair, was calculated using expected mean and expected standard deviation (std).

$${expected},{mean}=mu 1+mu 2$$

(1)

$${expected},{stand},{deviat}=sqrt{{({std}1)}^{2}+{({std}2)}^{2}}$$

(2)

$${S}_{{pooled}}=frac{sqrt{{({expected} , {std})}^{2}+{({observed} , {std})}^{2}}}{2}$$

(3)

$${Cohe}{n}^{{prime} }{sD}=frac{{expected},{mean}-{observed},{mean}}{{S}_{{pooled}}}$$

(4)

Where 1 = mean LFC of gene1 constructs, 2 = mean LFC of gene2 constructs, std1 = standard deviation of LFC of gene1 constructs, and std2 = standard deviation of LFC of gene2 constructs.

In each cell line, the paralog pairs with dLFC<1 and Cohens D>0.8 were selected as hits. Cohens D>0.8 indicates large effect size between two groups, meaning that our expected and observed distribution of gRNAs are meaningfully separated. In total 388 paralog pairs were identified as hits across all the studies.

To identify the most consistent method in terms of hit identification, the Jaccard similarity coefficient of every pair of cell lines in each study was calculated by taking the ratio of intersection of hits over union of hits. For the studies that screened more than two cell lines, the final platform weight was the median of the calculated Jaccard coefficients of all pairs of cell lines.

$$Jleft(A,, Bright)=frac{left|Acap Bright|}{left|Acup Bright|}=frac{left|Acap Bright|}{left|A{{{{{rm{|}}}}}}+left|Bright|-{{{{{rm{|}}}}}}Acap Bright|}$$

(5)

To score paralog pairs, each hit was scored based on the cell lines in which it was identified as a hit; cell lines were weighted based on the platform weight described above. We defined the paralog score as the sum of platform weights of cell lines in which the paralog pair was identified as a hit minus the sum of platform weights of cell lines in which the paralog pair was assayed but not identified as a hit (a miss). The distribution of scores is shown in Fig.1. Gene pairs with paralog score > 0.25 and were identified as a hit in two or more studies were listed as candidate gold standard paralog synthetic lethals.

To construct an all-in-one vector for expression of both Cas12a and a guide array, we first swapped in puromycin resistance in place of blasticidin resistance from pRDA_174 (Addgene #136476). We then tested four locations for the insertion of a U6-guide expression cassette; notably this cassette needs to be oriented in the opposite direction of the primary lentiviral transcript to prevent Cas12a-mediated processing during viral packaging in 293T cells. The construct with the best-performing location, between the cPPT and the EF-1 promoter, was designed pRDA_550 (Addgene #203398). Synthesis of DNA and custom cloning was performed by Genscript.

An oligonucleotide pool consisting of 7 Essential and 7 Non-Essential gene crRNAs with their nearby DR, BsmBI recognition as well as overhang sequence was synthesized by Integrated DNA Technologies. The pool was amplified by asymmetric PCR followed by being assembled into PRDA_550 vector to acquire the designed library through NEBridge Golden Gate Assembly Kit (BsmBI-v2) (New England Biolabs). The assembled product was transformed into NEB Stable Competent E. coli (High Efficiency) cells and the plasmid DNA was purified using the PureLink Plasmid Purification Kit (Invitrogen). Three oligonucleotide pools were cloned separately and pooled together to acquire the final 7mer library. The library was sequenced to confirm uniform and complete library representation.

Human paralogs and percent identity data were imported from BioMart, which reports both AB and BA percent identity (these can differ if the two genes encode proteins of different lengths) Mean percent identity ((AB+BA)/2)and delta percent identity (|ABBA|) between paralogs were then calculated, and for the prototype library, paralogs with mean percent identity between 30% and 99% and delta percent identity <10% were selected (Supplementary Fig.5). Next, CCLE expression data was downloaded, and the mean and standard deviation of expression across all CCLE samples was calculated for each gene. Paralogs where both genes had mean expression>2 and stdev<1.5 were selected (i.e. constitutively expressed genes).

Finally, to identify and include paralog families of size > 2, we applied a difference from top paralog filter. For each gene A in the pool, we identified its top paralog B by max sequence identity. Then for each other candidate paralog C, we calculated the drop in sequence identity, ABAC (see distribution of drop % in Supplementary Fig.5). For the prototype library, we defined A,B,C as being in the same family if ABAC<10%.

For the final Inzolia library, we relaxed several of these filters. The delta percent identity filter and the expression variance filter were removed entirely, and the difference from top paralog filter was expanded to 20%. The mean expression filter was retained. These three filtering steps resulted in a total of 4435 paralog pairs included in the Inzolia pool library.

Oligonucleotide pools consisting of designed four-plex guide arrays were synthesized by Twist Bioscience. The prototype pool consists of 43,972 arrays targeting 19,687 single genes, 2082 paralog pairs, 167 paralog triples, and 48 paralog quads.

5-AATGATACGGCGACCACCGAcgtctcgAGATnnnnnnnnnnnnnnnnnnnnTAATTTCTACTATTGTAGATnnnnnnnnnnnnnnnnnnnnAAATTTCTACTCTAGTAGATnnnnnnnnnnnnnnnnnnnnTAATTTCTACTGTCGTAGATnnnnnnnnnnnnnnnnnnnnTTTTTTGAATggagacgATCTCGTATGCCGTCTTCTGCTTG-3.

Italic: primer sequence Bold: BsmBI restriction sequence. Overhang in CAPS. nnnnn: guide sequence Underlined: DR sequence.

The pool of guide arrays was PCR amplified using KAPA HiFi 2X HotStart ReadyMix (Roche) using 20ng of starting template per 25L reaction (primers are listed in Supplementary Data10) and the following conditions: denaturation at 95C for 3min, followed by 12 cycles of 20s at 98C, 30s at 60C, and 30s at 72C, followed by a final extension of 1min at 72C. The resulting amplicon was purified by the Monarch PCR & DNA Cleanup Kit (New England Biolabs) and cloned into the pRDA-550 vector by NEBridge Golden Gate Assembly Kit (BsmBI-v2) The product from assembly reaction was purified and electroporated into Endura Electrocompetent cells (Lucigen). Transformed bacteria were diluted 1:100 in 2xYT medium containing 100g/mL carbenicillin (Sigma) and grown at 30 C for 16h. The plasmid DNA was extracted by PureLink Plasmid Purification Kit (Invitrogen). The library was sequenced to confirm uniform and complete library representation. The library was prepared in MD Anderson Cancer Center.

The final Inzolia pool consists of arrays targeting 19,687 single genes, 4435 paralog pairs, 376 paralog triples, and 100 paralog quads, plus 20 arrays targeting EGFP, 500 targeting intergenic loci, and 50 encoding non-targeting guides. Each array in the oligonucleotide pools is constructed as follows:

5-AGGCACTTGCTCGTACGACGcgtctcgAGATnnnnnnnnnnnnnnnnnnnnTAATTTCTACTATTGTAGATnnnnnnnnnnnnnnnnnnnnAAATTTCTACTCTAGTAGATnnnnnnnnnnnnnnnnnnnnTAATTTCTACTGTCGTAGATnnnnnnnnnnnnnnnnnnnnTTTTTTGAATggagacgTTAAGGTGCCGGGCCCACAT-3.

Italic: primer sequence Bold: BsmBI restriction sequence. Overhang in CAPS. nnnnn: guide sequence Underlined: DR sequence.

The pool of guide arrays was PCR amplified using NEBNext High-Fidelity 2X PCR Master Mix (NEB) using 196ng of starting template per 50L reaction (primers are listed in Supplementary Data10) and the following conditions: denaturation at 98C for 1min, followed by 7 cycles of 30s at 98C, 30s at 53C, and 30s at 72C, followed by a final extension of 5min at 72C. The resulting amplicon was purified by the Qiaquick PCR Purification Kit (Qiagen) and cloned into the pRDA-550 and pRDA-052 via Golden Gate cloning with Esp3I (Fisher Scientific) and T7 ligase (Epizyme). The assembly product was purified by isopropanol precipitation, electroporated into Stbl4 electrocompetent cells (Life Technologies) and grown at 37 C for 16h on agar with 100 ug/mL carbenicillin. Colonies were scraped and plasmid DNA (pDNA) was extracted via HiSpeed Plasmid Maxi (Qiagen). The library was sequenced to confirm uniform and complete library representation. The library was prepared in Broad institute.

K-562 and A549 cells were a gift from Tim Heffernan. A375 and MELJUSO were obtained from the Cancer Cell Line Encyclopedia. Cell line identities were confirmed by STR fingerprinting by M.D. Anderson Cancer Centers Cytogenetic and Cell Authentication Core. All cell lines were routinely tested for mycoplasma contamination using cells cultured in non-antibiotic medium (PlasmoTest Mycoplasma Detection Assay, InvivoGen).

All cell lines were grown at 37C in humidified incubators at 5.0% CO2 and passaged to maintain exponential growth. For each cell line, the following medium and concentration of polybrene (EMD Millipore) and puromycin (Gibco) were used:

K-562: RPMI+10% FBS, 8g/mL, 2g/mL

A549: DMEM+10%FBS, 8g/mL, 2g/mL

A375: RPMI+10% FBS, 1g/mL, 1g/mL

MELJUSO: RPMI+10% FBS, 4g/mL, 1g/mL.

Lentivirus was produced by the University of Michigan Vector Core (prototype) or the Broad GPP (Inzolia). Virus stocks were not titered in advance. Transduction of the cells was performed at 1X concentration of virus with corresponding polybrene. Non-transduced cells were eliminated via selection puromycin dihydrochloride. The selection was maintained until all non-transduced control cells reached 0% viability. Once selection with puromycin was complete, surviving cells were pooled and 500x coverage cells were harvested for a T0 sample. After T0, cells were harvested at 500X coverage on corresponding days. The prototype In4mer screens were performed in MD Anderson Cancer Center. The Inzolia screens were performed in Broad Institute.

Genomic DNA (gDNA) was extracted using the Mag-Bind Blood & Tissue DNA HDQ 96 Kit (Omega Bio-tek) and quantified by the Qubit dsDNA Quantification Assay Kits (ThermoFisher). Illumina-compatible guide array amplicons were generated by amplification of the gDNA in a one-step PCR. Indexed PCR primers were synthesized by Integrated DNA Technologies using the standard 8nt indexes from Illumina (D501-D508 and D701-D712) (Supplementary Data10).

At least ~200X coverage gDNA per replicate across multiple reactions were amplified. Each gDNA sample was first divided into multiple 50L reactions with most 2.5ug gDNA per reaction. Each reaction contained 1ul each primer (10M), 1L 50X dNTPs, 5% DMSO, 5L 10X Titanium Taq Buffer, and 1L 10X Titanium Taq DNA Polymerase (Takara). The PCR conditions were: denaturation at 95C for 60s, followed by 25 cycles of 30s at 95C and 1min at 68C, followed by a final extension at 68C for 3min. After the PCR, all reactions from the same sample were pooled and then purified by E-Gel SizeSelect II Agarose Gels, 2% (ThermoFisher). Purified amplicons were quantified by Qubit dsDNA Quantification Assay Kits (ThermoFisher) and validated by D1000 ScreenTape Assay for TapeStation Systems (Agilent) (360bp for in4mer, 501bp for 7Mer). Purified amplicons were then pooled (with 30% customized random library to increase the diversity) and sequencing was performed by NextSeq 500 sequencing platform (Illumina) with custom primers (Integrated DNA Technologies) (Supplementary Data10). The In4mer library was sequenced by read format of 151-8-8, single-end and the 7Mer library was sequenced by read format of 151-8-8-151, paired-end.

Genomic DNA (gDNA) was extracted using the Mag-Bind Blood & Tissue DNA HDQ 96 Kit (Omega Bio-tek) and quantified by the Qubit dsDNA Quantification Assay Kits (ThermoFisher). Illumina-compatible guide array amplicons were generated by amplification of the gDNA in a one-step PCR. Indexed PCR primers were synthesized by Integrated DNA Technologies using the standard 8nt indexes from Illumina (D501-D508 and D701-D712). The sequences for the primer sets were listed in Supplementary Data10.

At least ~200X coverage gDNA per replicate across multiple reactions were amplified. Each gDNA sample was first divided into multiple 100L reactions with most 10g gDNA per reaction. Each reaction contained 0.5L forward primer (100M), 10uL reverse primer (5 uM) 8L dNTPs, 5L DMSO, 10L 10X Titanium Taq Buffer, and 1.5L Titanium Taq DNA Polymerase (Takara). The PCR conditions were: An initial denaturation at 95C for 60s, followed by 28 cycles of 30s at 94C, 30s at 52C, and 30s at 72 C followed by a final extension at 72C for 10min. After the PCR, all reactions from the same sample were pooled and purified with Agencourt AMPure XP SPRI beads according to the manufacturer protocol (Beckman Coulter). Purified amplicons were quantified by Qubit dsDNA Quantification Assay Kits (ThermoFisher) and sequenced on a HiSeq2500 with a Rapid Run (200 cycle) kit (Illumina).

Reads for each reagent were counted using only exact matches to the entire 281 nucleotide 7mer sequence, excluding the leading DR (7 23mer spacer sequences + 6 20mer DR sequences). Fold changes were calculated relative to the mean of the T0 samples, and averaged across replicates. For each sample (T7/14/21), fold changes were normalized by subtracting the mean fold change of arrays with 7 nonessentials; i.e. setting no-essentials guides to zero.

We expected that the selected essential genes would not show any pairwise or higher order interactions, and thus should be governed by the multiplicative model of genetic interaction. To evaluate this model, we fit a regression model:

where A is a binary matrix of 7mer guide arrays (rows, k=384) by positions (columns, n=7), with Ai,j=1 if guide array i targets an essential gene at position j and 0 if not. (y) is the vector of normalized observed fold changes, and the n-length vector (beta) coefficients represent the single gene knockout phenotype learned from the model. We filtered this construct for reagents that encoded two or fewer essential genes (k=87 rows). After linear fit, we compared the predicted zero, one, and two gene knockout fitness profiles (by summing the (beta) coefficients for each gene) to the mean observed knockout fitness. R2 values for each pool ranged from 0.78 to 0.91, and the overall quality of the linear fit supports the multiplicative model for non-interacting genes as assayed by combinatorial CRISPR knockouts of up to two genes. An accurate null model for noninteraction is critical for detecting and classifying deviations from this model that reflect positive or negative genetic interactions.

In4mer library sequencing reads were mapped to the library using only perfect matches. BAGEL2 was used to normalize sample level read counts and to calculate fold changes relative to the T0 reference using the BAGEL2.py fc option with default parameters44. Essential and non-essential genes were defined using the Hart reference sets from refs. 39,41. Since the library targets both individual genes and specific gene sets (paralogs), we calculated the average gene/gene set (hereafter gene) log fold change as the mean of the clone-level fold changes across two replicates. All fold changes are calculated in log2 space. Cohens D statistics were calculated in Python as described in Paralog meta-analysis above. Data for recall-precision curves were calculated using BAGEL2. We set an arbitrary threshold of fc<1 for essential genes.

For genetic interaction analysis, the expected fold change was calculated as the sum of the gene-level fold changes for each individual gene in the gene set. Expected fc was subtracted from observed fc to calculate delta log fold change, dLFC, where negative dLFC indicates synthetic/synergistic interactions with more severe negative phenotype, and positive dLFC indicates positive/suppressor/masking interactions with less severe negative or more positive phenotype than expected. We set an arbitrary threshold of dLFC<1 for synthetic lethality, and>+1 for masking/suppressor interactions.

An arrayed knockout apoptosis assay approach was adopted to validate RAS synthetic lethality in K-562. Two guides were selected for each of the three RAS genes, and two clones were designed for each target/gene combination. Guide RNAs were selected through CRISPick and gblocks (same construct as Inzolia library) were synthesized by Integrated DNA Technologies. The arrays were individually cloned into the pRDA_550 backbone and plasmids were validated by Sanger sequencing. The plasmids were then individually transfected to K-562 cells via the Neon Transfection System (Invitrogen). Each group was transfected with 2g of DNA per 2106 cells, using the recommended setting for K-562 electroporation with one pulse at 1000v, 50ms. Non-transfected cells were eliminated through puromycin selection, which was maintained until non-transfected control cells reached 0% viability. Triplicate wells were maintained after selection until the end of the experiment. Cell viability, total cell numbers, live cell size and dead cell size data were collected through reading Trypan Blue (Gibco) stained cells via Countess II FL (Thermo Fisher) at each passage until 9 days after puromycin selection, in line with Inzolia screen end point of 8 days in K-562 cells. Percent dead cells were normalized to negative control and one-way ANOVA was conducted to compare experimental groups against the negative control for statistical significance.

Further information on research design is available in theNature Portfolio Reporting Summary linked to this article.

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From an early age, Allison Keeler 05 (biotechnology) always knew she wanted to be a scientist. As an adult, that dream has come true as she is an assistant professor in the Department of Pediatrics at the University of Massachusetts (UMass) Chan Medical School and the lead researcher in the Keeler Lab within the Horae Gene Therapy Center.

Keeler applied early to RIT, out of her high school in eastern Pennsylvania. After visiting the campus and learning the university had one of the few biotechnology programs available, she knew getting hands-on experience in research at RIT was the best path for her future.

Keeler earned her bachelors degree in three years and was able to take advantage of a trip to the Galpagos Islands, where she realized she wanted to become an academic and a professor. After a research position at Duke University, she earned her Ph.D. in biomedical sciences from the UMass Medical School, where she now works. Mentoring and teaching in a lab environment has become her passion.

Basics that I learned at RIT and each of my experiences have shaped where I am now and what Im really passionate about, said Keeler.

Her background has led her to one of the most revolutionary medical fields today: gene therapy. This technology approaches disease differently, by attempting to change genetic makeups to prevent and treat disease instead of traditional treatments like medication and surgery.

In her lab, Keeler is learning about and understanding immune responses to gene therapy and engineering and developing new novel gene therapies for the treatment of different diseases.

The field is progressing rapidly. When she was a graduate student, there were no approved therapies. Now, there are many, with more being approved every year.

Its been really interesting to watch the field evolve, said Keeler. Its an exciting time in this field because several gene therapies have recently been approved.

The scientific area is familiar to the dean of RITs College of Science, Andr Hudson, who is repeatedly sought out as an expert in biochemistry and microbiology. His research interests are closely related to Keelers, as both are excited about the future of science as it relates to the human body and disease.

The work by Dr. Keeler and colleagues in this space is at the forefront of science and medicine, said Hudson. I am heartened that one of our College of Science alumni is helping to lead the charge.

Keeler never envisioned she would be running her own gene therapy lab when she stepped on RITs campus as a biotechnology major. But as science grows and evolves, more and more possibilities for careers in science exist. She encourages students to keep their minds open and to explore all opportunities.

I didnt even know about gene therapy when I was at RIT, said Keeler. But science continues to expand. Keep being curious, keep asking questions.

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Good morning! A crowdsourced Readout today, to be sure, with contributions from STATs Jason Mast, Elaine Chen, and Jonathan Wosen. We get into earnings from Novo Nordisk, Amgen, and Illumina, and see that ICER is not so enthused about a gene therapy from Sarepta.

Also: Wed like to know what you think of this newsletter. if you havent, could you fill outthis survey?

Novo Nordisk CEO Lars Fruergaard Jrgensen is standing by the cost of Ozempic and Wegovy, despite an ongoing Senate investigation into the companys pricing. In an earnings call yesterday, he said the drugs offer an attractive value proposition as theyre priced similarly to earlier, less effective iterations of this class of drugs.

Although he conceded that the number of people taking the drugs is to some degree putting strains on health care systems, STATs Elaine Chen writes, the full value of these diabetes and obesity drugs has yet to be realized. Ozempics list price is $969 monthly; Wegovys is $1,349. Executives on the call said that the net prices for these drugs have already come down, and will continue to do so as more people take it and as competition increases.

Read more.

The removal of small amounts of brain tissue from desperately ill patients, done as part of a Mount Sinai research project, triggered alarm bells at the FDA and has raised broader questions about the scientific and ethical justification for live-brain research. Journalist and STAT contributor Katherine Eban joins The Readout LOUD this week to discuss the findings of atwo-year investigation.

Also on this weeks episode, STATs Adam Feuerstein and Allison DeAngelis discuss Novartis effort to acquire MorphoSys, and the latest news on Eli Lilly and Novo Nordisks blockbuster obesity drugs with Elaine Chen.

Listen here.

From STATs Jason Mast:The drug-pricing watchdogs over at the Institute for Clinical and Economic Review, or ICER, often take a dim view of modern drug pricing exceptwhen it comes to genetherapy. Those treatments, the nonprofit has said, often provide the years-long benefits for severe disease that justify a multimillion-dollar price tag.

Which makes theJAMApiecepublishedthis week by ICER CMO David Rind all the more notable. Rind considered Elevidys, Sareptas $3.2 million gene therapy for Duchenne muscular dystrophy. The treatment was given accelerated approval last year for 4- and 5-year-olds and, after a Phase 3 trial, the FDA is now considering whether to approve it for all ages.

That Phase 3 trial missed its primary endpoint, though, as did a previous smaller study. Sarepta has pointed to the results on secondary measures but given those data, Rind cast doubt on whether it should be approved and certainly whether Sarepta should charge what other gene therapy companies do. This is an enormous price tag for a therapy that has failed to meet its primary end point in the 2 randomized trials in which it has been studied and that is clearly not curative, he wrote.

From STATs Jonathan Wosen:DNA sequencing juggernaut Illumina reported yesterday $1.06 billion in revenue for its core business during the first quarter of this year, down 2% from the same time last year, with the companys execs reiterating that they expect 2024 revenue to essentially match the $4.5 billion from the previous fiscal year.

CEO Jacob Thaysen cautiously described the first-quarter numbers as a decent start to the year that exceeded expectations on a call with market analysts and investors. The updated numbers come during a turbulent time for the broader sequencing space. Pacific Biosciences, a Bay Area company that Illumina once unsuccessfully tried to acquire, recently laid off nearly 200 employees after reporting disappointing first-quarter sales. And while Illumina continues to control about 80% of the market, its shares are down 37% from a year ago and the firm faces growing competition from players such asUltima, Singular Genomics, andElement Biosciences.

The San Diego genomics giant is still on track to finalize the terms of its divestiture of Grail a cancer detection startup Illumina had acquired for $8 billion, drawing the ire of regulators in the U.S. and Europe by the end of the second quarter of this year.

From STATs Elaine Chen:Amgen will scrap an early-stage obesity pill, and will instead focus on its more advanced injectable candidate called MariTide thats seen as a potential competitor to Wegovy and Zepbound.

MariTide is in a Phase 2 trial for obesity, and CEO Bob Bradway said on the earnings call yesterday that we are very encouraged with the results from an interim analysis of that trial. The company is planning a broad Phase 3 program that will study MariTide in obesity, diabetes, and obesity-related conditions, and its already gearing up to make large amounts of the drug, initiating activities to further expand manufacturing capacity with both clinical and commercial supply in mind, Bradway said.

MariTide has an interesting mechanism its a monoclonal antibody linked to two peptides that activates receptors of the GLP-1 hormone while blocking receptors of the GIP hormone. Even though that appears to contradictory to the mechanism of Lillys potent obesity drug Zepbound, which activates both GLP-1 and GIP receptors, MariTide has shown potential to induce potent and longer-lasting weight loss.

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Electrical engineering PhD student Yizhou Yao had to develop a whole new set of skills to research the effects of electric pulses on the receptivity of liver cells to new types of gene therapy. Photo by Joel Hallberg

In an effort to improve delivery of costly medical treatments, a team of researchers in electrical engineering at the University of WisconsinMadison has developed a stimulating method that could make the human body more receptive to certain gene therapies.

The researchers exposed liver cells to short electric pulses and those gentle zaps caused the liver cells to take in more than 40 times the amount of gene therapy material compared to cells that were not exposed to pulsed electric fields. The method could help reduce the dosage needed for these treatments, making them much safer and more affordable. The research appears April 30 in the journal PLOS ONE.

Gene therapy is a promising medical technology: By replacing, altering or introducing new genetic material into a patients cells, doctors may be able to cure or compensate for genetic diseases, including cystic fibrosis, sickle-cell disease, hemophilia and diabetes.

One of the bottlenecks in gene therapy, however, is getting the right dose of genetic material into the target cells. The UWMadison research suggests that applying a moderate electric field, which left no lasting damage to the cells that received it, could help in creating more effective therapies.

The project began almost a decade ago with Hans Sollinger, a world-renowned transplant surgeon at UWMadison. He had developed a gene therapy treatment for Type 1 diabetes, an autoimmune disease that attacks the pancreas, the organ that produces insulin.

From left, Professor John Booske, PhD student Yizhou Yao and Professor Susan Hagness, all in the UWMadison Department of Electrical and Computer Engineering. Photo by Joel Hallberg

Sollingers treatment strategy delivered the genetic code for insulin production into liver cells using an adreno-associated virus that assists in transporting the therapeutic genes across the cells membrane. This DNA can then take up residence in liver cells, producing insulin without being attacked by the immune system in the pancreas.

While Sollinger had a proof of concept that the therapy worked, he believed the future of the treatment hinged on delivery. He turned to Susan Hagness and John Booske, both UWMadison professors of electrical and computer engineering who have experience treating human cells with electrical pulses.

What we started talking about was local, targeted delivery and whether there was a way of getting the treatment DNA directly into the liver without passing it through the entire body and triggering the immune system, says Hagness. And whether we could use electric pulses in order to make this delivery process more efficient and dramatically reduce the dose needed.

Researchers have previously found that exposing cells to electric fields can often increase the ability of molecules to move through the cell membrane into the interior of a cell. So, in this latest study, PhD student Yizhou Yao sought to determine whether the technique would increase the penetration of virus particles into liver cells.

Using human hepatoma cells, a model system for studying the liver, Yao exposed batches of the cells to various concentrations of the gene therapy virus particles containing a fluorescent green protein. She used a pair of electrodes to deliver an 80-millisecond electric pulse to some samples, then incubated all the cells for 12 hours.

When she examined the results 48 hours later under a fluorescence microscope, Yao found that only a small percentage of the cells that had not received the electrical pulses glowed green. In stark contrast, those cells that had received a zap accumulated about 40 times the amount of the fluorescent green proteins delivered by the virus.

While results provided compelling evidence that the pulses helped facilitate the viruss penetration of the cell walls, Booske says the team has yet to discover exactly how the process works at the molecular level.

Theres enough known about electric pulsing that I think we could confidently state that it is opening nanopores through the cell membrane, he says. But then Yao got this remarkable result, and it dawned on us that virus particles are in general bigger and more complex than bare molecular particles and they already have their own way of getting inside cells. So, we dont really know if its the pores opening that has anything to do with it directly or indirectly.

Sollinger passed away in May 2023, but the team says his legacy will live on through the ongoing research on this project and the work of other groups. The electrical engineering researchers are pursuing next steps with external funding and are optimistic that ultimately the technique will translate into clinical trials.

Yao, who will graduate in 2024, says she knew the study would be transdisciplinary, but didnt realize just how far it would go.

I am an electrical engineer by training, and I dont have a biology background, she says. Before this, the last time I used a microscope was in high school. It was quite a steep learning curve, learning to culture cells and carry out biology protocols. But I really enjoyed this project and liked its ultimate goal, which is to make the world a better place.

Other authors include Robert W. Holdcraft of the Cincinnati Childrens Hospital Medical Center.

Original post:
An electrifying discovery may help doctors deliver more effective gene therapies - University of Wisconsin-Madison

Recommendation and review posted by Bethany Smith

SMN-K186R, a modified version of the SMN protein that is deficient in people with spinal muscular atrophy (SMA), was more effective than normal SMN when delivered by gene therapy, a mouse study demonstrated.

Treatment with lower doses of the modified SMN gene therapy, to reduce liver-related toxicity seen with current gene therapies, was still more effective than normal SMN.

[SMN-K186R] has value as a new treatment for SMA that improves treatment effectiveness and reduces adverse events simultaneously, the scientists wrote.

The study, Improved therapeutic approach for spinal muscular atrophy via ubiquitination-resistant survival motor neuron variant, was published in the Journal of Cachexia, Sarcopenia and Muscle.

SMA is caused by defects in the SMN1 gene, which leads to a deficiency in the SMN protein. A lack of SMN mainly affects motor neurons, the specialized nerve cells that control movement, resulting in muscle weakness and atrophy (shrinkage) over time.

Zolgensma (onasemnogene abeparvovec-xioi) is a gene therapy widely approved for newborns and toddlers up to age 2 with all types of SMA. Delivered to cells using a modified and harmless adeno-associated virus serotype 9 (AAV9), the therapy is designed to replace the faulty SMN1 gene and increase the levels of SMN protein.

Although clinical trials have shown that Zolgensma can improve motor skills and extend survival, some patients still fail to achieve motor milestones despite treatment before symptom onset. Moreover, in clinical trials some patients showed liver toxicity due to high AAV9 dosage, even after treatment with immunosuppressants.

Therefore, drug development for an improved therapeutic effect for SMA patients is still needed, the scientists wrote.

In cells, proteins are routinely produced and then degraded in an ongoing dynamic process, depending on cellular needs. Proteins targeted for degradation are tagged with a small protein called ubiquitin, a process called ubiquitination.

In previous work, the research team created a mutant version of the SMN protein, called SMN-K186R, that was resistant to ubiquitination but still retained its function in motor neurons. As a result, the levels of SMN-K186R were sustained for longer periods compared with normal SMN (SMN-WT).

Researchers have now developed an AAV9-based gene therapy, similar to Zolgensma, that delivers the genetic instructions for SMN-K186R to cells and tested its efficacy in a mouse model of severe SMA.

Control SMA mice treated with a mock therapy had an average lifespan of 14 days and survived about four weeks following treatment with SMN-WT. In contrast, mice given SMN-K186R lived an average of 164 days, representing an average lifespan extension of more than 140 days, about 10 times longer than without treatment.

Body weights paralleled the survival results. Untreated mice, or those treated with SMN-WT, initially gained weight and then lost weight until death, whereas mice given SMN-K186R gained weight and size comparable to healthy mice.

Tissue analysis found higher levels of SMN protein in the brain, spinal cord, and muscles in SMA mice treated with SMN-K186R compared with SMN-WT, a result related to ubiquitination resistance. Higher SMN levels due to SMN-K186R coincided with more motor neurons in the spinal cord, greater muscle mass, and thicker muscle fibers.

Several motor function tests showed the improved efficacy of SMN-K186R over SMN-WT. In response to gravity, mice treated with SMN-K186R turned their bodies upward successfully, unlike SMN-WT-treated mice, which failed to turn upwards beyond day 23. Hind-limb clasping upon tail suspension occurs in mice with motor deficits: whereas mice treated with SMN-K186R rarely showed this behavior, SMN-WT-treated mice displayed hind-limb clasping for more than 20 out of 30 seconds.

Due to the potential of treatment-related liver failure (hepatotoxicity) associated with gene therapy, the team examined the effects of SMN-K186R on the liver. By day 25, the average liver weight of mice treated with SMN-K186R was within the normal range, whereas SMN-WT-treated mice had significantly lighter livers.

While the number of liver cells was similar between the two groups, only mice treated with SMN-K186R showed liver cell growth comparable to that of healthy mice. Moreover, liver cells from SMN-WT-treated mice showed signs of SMN protein aggregation, which has been reported to cause defects in motor neurons.

When the dose of SMN-K186R was lowered to one-third of the clinical viral dose to reduce liver toxicity, SMN-WT-treated mice displayed hind-limb clasping, whereas mice treated with the lower dose of SMN-K186R showed no signs of motor deficits.

The higher stability and levels of SMN-K186R generated over SMN-WT did not cause neurotoxicity in the brain and spinal cord or showed early signs of neurotoxicity when delivered directly into the bloodstream.

Our research suggests that an improved therapeutic approach for SMA via ubiquitination-resistant SMN, [SMN-K186R], will achieve better therapeutic effects in severe SMA newborn patients, the scientists wrote. The enablement of low AAV dose treatment from the improved treatment effects of [SMN-K186R] provides strong foundations for clinical applications in SMA patients to reduce hepatotoxicity.

Read more here:
Modified SMN protein aids gene therapy efficacy: Mouse study - SMA News Today

Recommendation and review posted by Bethany Smith

LONDON In 2021, a team of Italian researchers reported that an experimental gene therapy they had developed seemed to be correcting the metabolic issues at the core of a rare genetic disease that left children unable to break down sugar molecules.

There remained an open question, however, about whether the therapy could address a particularly debilitating manifestation of the disease, the severe skeletal deformities it caused. Patients with the disease, called Hurler syndrome, suffer from short stature, spinal defects, and extremely stiff joints, complications that greatly limit their quality of life.

But in a new paper, the research team showed that the gene therapy, when delivered to toddlers, successfully staved off those problems for years. The children grew to heights within average norms, and had far more flexible shoulder, hip, and knee joints than untreated children, according to the study, published Wednesday in the journal Science Translational Medicine. Defects in the patients spines also stabilized.

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Experimental gene therapy seems to alleviate skeletal defects tied to rare inherited disease, study shows - STAT

Recommendation and review posted by Bethany Smith

Summary: Researchers made a significant breakthrough in ALS treatment using a new gene therapy, marking a notable slow in disease progression for a patient with an aggressive form of ALS. The patient, treated since early 2020, has maintained much of their physical and social abilities, exceeding typical life expectancy and functionality projections for their condition.

This therapy targets the SOD1 gene mutation, reducing levels of a harmful protein and stabilizing the patients condition. The findings offer hope for future advancements in ALS treatment and broader applications of gene therapy.

Key Facts:

Source: Umea University

There has been a breakthrough in the research on the disease amyotrophic lateral sclerosis (ALS).

Scientists at Ume University report that the disease progression in a patient with a particularly aggressive form of ALS disease has slowed down considerably with the use of a new gene therapy.

After four years on the medication, the patient can still climb stairs, rise from his chair, eat and speak well, and live an active and socially fulfilling life.

I consider this a breakthrough for the research we have conducted for more than 30 years, here at Ume University and University Hospital of Northern Sweden. We have never before seen treatment results as effective as these, using any other treatment, says Peter Andersen, a neurologist and professor at the Department of Clinical Sciences at Ume University.

An important discovery is that it is now possible to considerably reduce the levels of the disease-causing SOD1 protein, and simultaneously measure a clear inhibitory effect on furtherdisease progression.

When we diagnosed the patient at the neurology ward inearly spring2020, the patients prognosis was 1.52 years of survival at best. The patient has far, far exceeded expectation.

The patient is from a family in southern Sweden with a particularly aggressive form of ALS disease caused by a mutation in the SOD1 gene. When a relative was diagnosed with ALS, the patient left ablood samplefor research purposes to the ALS research team at Ume University but chose to not learn about the results of the genetic test.

However, the patient was a carrier of the disease gene, and after experiencing muscle weakness four years ago, the patient realized that he too was afflicted. The patient was immediately received by the medical team at University Hospital of Northern Sweden and was diagnosed with early stage ALS disease.

Since the summer of 2020, the patient has been a participant in the Phase III study evaluating a new gene therapy developed for patients with SOD1 mutations causing misfolding and aggregation of SOD1 protein in motorneurons.

Every four weeks, the patient received theexperimental treatmentat a university hospital in Copenhagen in Denmark.

At the time of diagnosis in 2020, the patients levels of the substance neurofilament La biomarker indicating breakdown of nerve cellswas very high. Now, four years later, the levels are reduced by almost 90%.

When the patient was diagnosed at University Hospital of Northern Sweden in April 2020, we measured the level of neurofilament L to be as high as 11,000 nanograms per liter, which is high even for an ALS patient.

In the most recent sample, after 50 injections of the new drug, the level is down to 1,200 to 1,290, which is a substantial decrease of the disease indicator, says Peter Andersen.

The normal level for a person in the patients age group is below 560. In blood, the level of neurofilament has fallen back to normal levels, and was down to 12 during the latest hospital visit. The normal level is less than 13.

The patients level of function, measured using the scale ALSFRSR, is reduced compared to a healthy individual (48 points) but has stayed at almost the same level, around 35 to 37 points, for the last 18 monthsthat means that the patients functional level is reduced by approximately 26% compared to a healthy individual.

A person with this aggressive type of ALS gene mutation that the patient has typically loses 11.5 points every month. That means that without treatment, the expected disease progression would have been very fast and given rise to substantial disability within 612 months, and, most likely, have lead to the patients death in 2021.

That this patient, more or less unimpeded, still can climb stairs four years after disease onset, that is somewhat of a miracle to see, says Karin Forsberg, a neurologist and researcher at the Department of Clinical Sciences who works alongside Peter Andersen and has researched SOD1 and ALS for more than two decades.

To have succeeded with adrug treatmentin this way is a great success and an inspiration. But it does not in any way mean that the job is done. This is just the beginning.

It is also important to remember that the drug in question does not constitute a curative treatment, but it seems able to put the brake on disease progression. It gives us great hope to further develop pharmaceutical treatments for ALS-patients.

There are many types of ALS disease, and only 2% to 6% has an ALS disease caused by a mutation in the SOD1 gene. Many have a familial form of the disease, but mutations in SOD1 have also been found in so-called sporadic cases of ALS.

Whether this drug has a similar effect on other types of ALS disease is currently unknown. There is need for much more research on the subject, says Peter Andersen.

The patient can still do almost all things that he could do when he first joined the study in the summer of 2020his speech is unaffected, and he manages to do everything himself, he mows the lawn, goes shopping, and takes care of his children. Mentally he also feels a lot better, mainly because he now dares to feel hope.

The study that the patient is participating in ends this summer. The medication is not yet available in Sweden, but it has been approved by the United States Food and Drug Administration, FDA, and on the 23 of February 2024 the European Medicines Agency, EMA, recommended the use of the drug on patients with SOD1 gene mutations within the European Union.

However, the New Therapies Council i Sweden has asked the regional health care providers not to prescribe the drug until a health economic evaluation has been provided by the Dental and Pharmaceutical Benefits Agency.

Our next step is to study the results from the patients receiving this drug. It has worked for some, but not all have seen the same positive effect. It could be a question of dosage, or at which disease stage the treatment was initiated.

Maybe additional drugs are required to completely stop the process? Those are questions we now have to try and answer. This is only the beginning, says Karin Forsberg.

She pictures a future where treatment will be given based on what type of ALS disease the patient has, and that it most likely will require a combination of drugs.

She emphasizes that there is much research being conducted both in Sweden and internationally to find new drug targets so that equivalent drugs can be developed for patient groups with other types of ALS, and she is hopeful that it will come true.

We can measure in samples collected from the patient that the disease process is ongoing, but the patients body seems able to compensate. Even now, four years after the patient started taking this new gene therapy drug.

The Swedish Ethical Review Authority approved participation in these studies and now, several years later, we, as well as ALS physicians in other participating countries, see a clear clinical effect on many treated patients, says Peter Andersen.

The next step will be to get approval from the Swedish Ethical Review Authority to study the compensatory mechanisms that treatment with this drug seems to have activated. There might be an opportunity here to get insights into how previously unknown parts of the nervous system work, and to develop even better new drugs.

Author: Peter Andersen Source: Umea University Contact: Peter Andersen Umea University Image: The image is credited to Neuroscience News

Original Research: The findings will appear in eLife.

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Gene Therapy Slows ALS Progression - Neuroscience News

Recommendation and review posted by Bethany Smith

Poseida Therapeutics and Astellas, under its Xyphos Biosciences subsidiary, have initiated a research collaboration and licensing agreement to develop allogeneic cell therapy programs by combining their innovative cell therapy platforms and technologies.

The companies plan to combine Poseida's proprietary allogeneic chimeric antigen receptor T-cell (CAR-T) platform with Xyphos' Accel platform (which combines its convertibleCAR and proprietary MicAbodies to target tumor cells) to create a Poseida-developed CAR-T construct. This CAR-T construct will then be used to develop two convertibleCAR product candidates targeting solid tumors.

Under the terms of the agreement, Poseida will receive $50 million upfront, plus potential development and sales milestones and contingency payments of up to $550 million in total. Additionally, Poseida is eligible for royalties as a percentage of net sales.

Xyphos will reimburse Poseida for costs incurred as part of the research agreement and will be responsible for the development and future commercialization of the products generated from the collaboration.

In August 2023, Astellas invested $25 million for an 8.8% stake in Poseida and paid an additional $25 million for the right to exclusive negotiation and first refusal to license Poseidas P-MUC1C-ALLO1 CAR-T cell therapy product candidate.

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Astellas and Poseida to collaborate on oncology cell therapies - LabPulse

Recommendation and review posted by Bethany Smith

Research Triangle Park, N.C., May 02, 2024 (GLOBE NEWSWIRE) -- Asklepios BioPharmaceutical, Inc. (AskBio), a gene therapy company wholly owned and independently operated as a subsidiary of Bayer AG, will deliver nine presentations offering insights into its adeno-associated virus (AAV) research and development, a key area of gene therapy focus for the company, at the American Society of Gene and Cell Therapy (ASGCT) 27th Annual Meeting, which takes place May 711, 2024, in Baltimore, Maryland, USA. Presentations will focus on AAV immune-mediated responses as well as the results from the ongoing Phase 1 clinical trial of AB-1002 gene therapy in patients with advanced heart failure.

Luke Roberts, MBBS, PhD, Medical Director for Clinical Development at AskBio, will deliver an oral presentation sharing new clinical data from the companys ongoing Phase 1 trial of AB-1002 in patients with advanced heart failure. This follows AskBios recent news that AB-1002 was granted FDA Fast Track Designation for the treatment of congestive heart failure (CHF). AB-1002 (also known as NAN-101) is an investigational gene therapy that has not yet received marketing authorization, and its efficacy and safety have not been established or fully evaluated. AskBio previously communicated that the delivery of AB-1002 was well tolerated and resulted in positive preliminary efficacy outcomes in some patients with non-ischemic CHF and may validate that the AAV2i8 vector capsid used is highly cardiotropic when administered as a single intracoronary infusion at relatively low doses.

Preliminary data from the Phase 1 trial of AB-1002 were presented at the 2023 American Heart Association Scientific Sessions in November, and AskBio began enrolling patients in its Phase 2 GenePHIT study of AB-1002 in adults with non-ischemic cardiomyopathy and New York Heart Association (NYHA) Class III heart failure symptoms in January 2024.

AskBios ASGCT presence will also include key presentations showcasing the companys continued commitment to optimizing AAV as a gene therapy, with a focus on preventing or reducing AAV immune response-related adverse events, which remains a vital area of investigation across the gene therapy treatment landscape. Attendees can look forward to an ASGCT spotlight speaker presentation on current immune modulation strategies given by Shari Gordon, PhD, Senior Director of Immunology at AskBio, on Day 3. On Day 4, Shari Gordon will deliver on behalf of Audry Fernandez, PhD, Principal Scientist & Group Lead, Immunology R&D at AskBio, an oral presentation on pre-clinical research into AAV-specific immune responses, and Liujiang Song, PhD, Principal Scientist for R&D Capsid and Biology at AskBio, will offer insights into AAV biology and vector intracellular fate during an oral presentation on AAV episome configuration using third generation long-read sequencing technologies and advanced bioinformatics.

Our presence at ASGCT this year highlights our continued commitment to sharing AAV developments with the gene therapy community. Covering clinical and pre-clinical research, our presentations show our robust progress and ongoing ambition to bring to patients transformative therapies that were once unthinkable, said Gustavo Pesquin, Chief Executive Officer, AskBio. With our clinical and early-stage programs advancing, these are exciting times at AskBio.

With an ambitious portfolio of gene therapies at various stages of research and development, AskBio continues to develop AAV-based therapies to treat some the worlds most debilitating diseases, including CHF, Huntingtons disease, limb-girdle muscular dystrophy, multiple system atrophy, Parkinsons disease, and Pompe disease. By targeting these therapy areas, AskBio aims to deliver breakthrough treatments that could benefit more than an estimated 35 million patients worldwide.17

AskBios presentations at ASGCT include:

About AskBio

Asklepios BioPharmaceutical, Inc. (AskBio), a wholly owned and independently operated subsidiary of Bayer AG, is a fully integrated gene therapy company dedicated to developing life-saving medicines and changing lives. The company maintains a portfolio of clinical programs across a range of neuromuscular, central nervous system, cardiovascular, and metabolic disease indications with a clinical-stage pipeline that includes therapeutics for congestive heart failure, Huntingtons disease, limb-girdle muscular dystrophy, multiple system atrophy, Parkinsons disease, and Pompe disease. AskBios gene therapy platform includes Pro10, an industry-leading proprietary cell line manufacturing process, and an extensive capsid and promoter library. With global headquarters in Research Triangle Park, North Carolina, and European headquarters in Edinburgh, Scotland, the company has generated hundreds of proprietary capsids and promoters, several of which have entered pre-clinical and clinical testing. An early innovator in the gene therapy field, with over 900 employees in five countries, the company holds more than 800 patents and patent applications in areas such as AAV production and chimeric capsids. Learn more at http://www.askbio.com or follow us on LinkedIn.

About Bayer

Bayer is a global enterprise with core competencies in the life science fields of health care and nutrition. In line with its mission, Health for all, Hunger for none, the companys products and services are designed to help people and the planet thrive by supporting efforts to master the major challenges presented by a growing and aging global population. Bayer is committed to driving sustainable development and generating a positive impact with its businesses. At the same time, the Group aims to increase its earning power and create value through innovation and growth. The Bayer brand stands for trust, reliability and quality throughout the world. In fiscal 2023, the Group employed around 100,000 people and had sales of 47.6 billion euros. R&D expenses before special items amounted to 5.8 billion euros. For more information, go towww.bayer.com.

About Viralgen Vector Core

Viralgen is a Contract Development and Manufacturing Organization (CDMO) founded in 2017 and exists as an independently operated subsidiary of AskBio, which is wholly owned and independently operated as a subsidiary of Bayer AG. As a manufacturer of Current Good Manufacturing Practice (cGMP) certified AAV, Viralgen offers the Pro10 based suspension manufacturing platform, a technology licensed from AskBio and developed by Chief Technical Officer Josh Grieger, PhD, and Co-Founder R. Jude Samulski, PhD, at University of North Carolina. The Pro10 platform has been found to increase scalability, performance, and yield of AAV therapies.8 Located in Spain, in the Gipuzkoa Science and Technology Park, Viralgen produces AAV gene therapy treatments for pharmaceutical and biotech companies with the aim of accelerating the delivery of new treatments that may improve patients lives.

The companys clinical facilities have four cGMP manufacturing suites, with 250-liter and 500-liter bioreactors. In 2020, Viralgen expanded within the Scientific Park by constructing a new building with three modules for large-scale commercial manufacturing. Each module of the state-of-the-art space includes three cGMP suites with a manufacturing capacity of >2,000 liters. The first module, which includes a suite dedicated to fully automated fill and finish operations, has received cGMP certification by the Spanish Agency for Medicines and Medical Devices (AEMPS) as part of the EMA network. For more information, visit viralgenvc.com.

Bayer Forward-Looking Statements

This release may contain forward-looking statements based on current assumptions and forecasts made by Bayer management. Various known and unknown risks, uncertainties and other factors could lead to material differences between the actual future results, financial situation, development or performance of the company and the estimates given here. These factors include those discussed in Bayers public reports which are available on the Bayer website atwww.bayer.com. The company assumes no liability whatsoever to update these forward-looking statements or to conform them to future events or developments.

AskBio Forward-Looking Statements

This press release contains forward-looking statements. Any statements contained in this press release that are not statements of historical fact may be deemed to be forward-looking statements. Words such as believes, anticipates, plans, expects, will, intends, potential, possible, and similar expressions are intended to identify forward-looking statements. These forward-looking statements include, without limitation, statements regarding AskBios clinical trials. These forward-looking statements involve risks and uncertainties, many of which are beyond AskBios control. Known risks include, among others: AskBio may not be able to execute on its business plans and goals, including meeting its expected or planned clinical and regulatory milestones and timelines, its reliance on third-parties, clinical development plans, manufacturing processes and plans, and bringing its product candidates to market, due to a variety of reasons, including possible limitations of company financial and other resources, manufacturing limitations that may not be anticipated or resolved in a timely manner, potential disagreements or other issues with our third-party collaborators and partners, and regulatory, court or agency feedback or decisions, such as feedback and decisions from the United States Food and Drug Administration or the United States Patent and Trademark Office. Any of the foregoing risks could materially and adversely affect AskBios business and results of operations. You should not place undue reliance on the forward-looking statements contained in this press release. AskBio does not undertake any obligation to publicly update its forward-looking statements based on events or circumstances after the date hereof.

References

[1] Balestrino R, Schapira AHV. Parkinson disease. Eur J Neurol. 2020;27(1):27-42.

[2] World Health Organization. Parkinson Disease. Available at:https://www.who.int/news-room/fact-sheets/detail/parkinson-disease. Accessed April 2024.

[3] Medina A, et al. Prevalence and Incidence of Huntington's Disease: An Updated Systematic Review and Meta-Analysis. Mov Disord. 2022;37(12):2327-2335.

[4] Malik A, et al. Congestive Heart Failure. In: StatPearls. Treasure Island (FL): StatPearls Publishing; November 7, 2022.

[5] MedlinePlus Genetics. NIH. Limb-girdle muscular dystrophy. Available at:https://medlineplus.gov/genetics/condition/limb-girdle-muscular-dystrophy/#frequency. Accessed April 2024.

[6] Goh Y, et al. Multiple system atrophy [published online ahead of print, 2023 Mar 16]. Pract Neurol. 2023; practneurol-2020-002797.

[7] Stevens D, et al. Pompe Disease: a Clinical, Diagnostic, and Therapeutic Overview. Curr Treat Options Neurol. 2022;24(11):573-588.

[8] Grieger JC, Soltys SM, Samulski RJ. Production of Recombinant Adeno-associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP FIX and FLT1 Clinical Vector. Mol Ther. 2016;24(2):287-297.

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AskBio Announces Nine Presentations at American Society of Gene and Cell Therapy 27th Annual Meeting 2024 - GlobeNewswire

Recommendation and review posted by Bethany Smith

SOLANA BEACH, Calif., May 02, 2024 (GLOBE NEWSWIRE) -- ClearPoint Neuro, Inc. (Nasdaq: CLPT) (the Company), a global device, cell, and gene therapy-enabling company offering precise navigation to the brain and spine, today announced it will present novel research and exhibit at the 27th American Society of Gene & Cell Therapy Annual Meeting to be held in Baltimore, MD from May 7-11.

The following original research abstracts will be presented at the Poster Session:

Additionally, the Companys technology will be featured in multiple partner posters and presentations. Conference attendees may visit the ClearPoint Neuro booth, #1033, to obtain a comprehensive list. Attendees may also pre-book meetings with our team directly here.

The ASGCT Annual Meeting provides an extraordinary opportunity for us to connect with our existing partners, and attract potential new customers, stated Jeremy Stigall, Chief Business Officer at ClearPoint Neuro. This year, in addition to the posters listed above, there are more than 10 partner-authored abstracts that are enabled by ClearPoint Neuro innovations.

About ClearPoint Neuro

ClearPoint Neuro is a device, cell, and gene therapy-enabling company offering precise navigation to the brain and spine. The Company uniquely provides both established clinical products as well as pre-clinical development services for controlled drug and device delivery. The Companys flagship product, the ClearPoint Neuro Navigation System, has FDA clearance and is CE-marked. ClearPoint Neuro is engaged with healthcare and research centers in North America, Europe, Asia, and South America. The Company is also partnered with the most innovative pharmaceutical/biotech companies, academic centers, and contract research organizations, providing solutions for direct CNS delivery of therapeutics in pre-clinical studies and clinical trials worldwide. To date, thousands of procedures have been performed and supported by the Companys field-based clinical specialist team, which offers support and services to our customers and partners worldwide. For more information, please visit http://www.clearpointneuro.com.

Forward-Looking Statements

This press release contains forward-looking statements within the context of the federal securities laws, which may include the Companys expectations for the future performance, market, and revenue of its products and services. These forward-looking statements are based on managements current expectations and are subject to the risks inherent in the business, which may cause the Company's actual results to differ materially from those expressed in or implied by forward-looking statements. Particular uncertainties and risks include those relating to: global and political instability, supply chain disruptions, labor shortages, and macroeconomic and inflationary conditions; future revenue from sales of the Companys products and services; the Companys ability to market, commercialize and achieve broader market acceptance for new products and services offered by the Company; the ability of our biologics and drug delivery partners to achieve commercial success, including their use of the Companys products and services in their delivery of therapies; the Companys expectations, projections and estimates regarding expenses, future revenue, capital requirements, and the availability of and the need for additional financing; the Companys ability to obtain additional funding to support its research and development programs; the ability of the Company to manage the growth of its business; the Companys ability to attract and retain its key employees; and risks inherent in the research, development, and regulatory approval of new products. More detailed information on these and additional factors that could affect the Companys actual results are described in the Risk Factors section of the Companys Annual Report on Form 10-K for the year ended December 31, 2023, which has been filed with the Securities and Exchange Commission, and the Companys Quarterly Report on Form 10-Q for the three months ended March 31, 2024, which the Company intends to file with the Securities and Exchange Commission on or before May 15, 2024. The Company does not assume any obligation to update these forward-looking statements.

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ClearPoint Neuro to Present Novel Research and Exhibit at the 27th American Society of Gene & Cell Therapy Annual ... - GlobeNewswire

Recommendation and review posted by Bethany Smith

San Diego, USA-based clinical-stage cell and gene therapy company Poseida Therapeutics (Nasdaq: PSTX) saw its shares close up more than 20% at $2.92 yesterday, when it revealed a second collaboration with Japanese drug major Astellas Pharma (TYO: 4503).

Astellas wholly-owned Xyphos Bioscience and Poseidahave entered into a research collaboration and license agreement to develop novel convertible CAR programs by combining the innovative cell

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Original post:
Astellas and Poseida enter second research collaboration - The Pharma Letter

Recommendation and review posted by Bethany Smith