Preinfusion polyfunctional anti-CD19 chimeric antigen receptor T cells are associated with clinical outcomes in NHL.

After treatment with chimeric antigen receptor (CAR) T cells, interleukin-15 (IL-15) elevation and CAR T-cell expansion are associated with non-Hodgkin lymphoma (NHL) outcomes. However, the association of preinfusion CAR product T-cell functionality with clinical outcomes has not been reported. A single-cell analysis of the preinfusion CD19 CAR product from patients with NHL demonstrated that CAR products contain polyfunctional T-cell subsets capable of deploying multiple immune programs represented by cytokines and chemokines, including interferon-γ, IL-17A, IL-8, and macrophage inflammatory protein 1α. A prespecified T-cell polyfunctionality strength index (PSI) applied to preinfusion CAR product was significantly associated with clinical response, and PSI combined with CAR T-cell expansion or pretreatment serum IL-15 levels conferred additional significance. Within the total product cell population, associations with clinical outcomes were greater with polyfunctional CD4+ T cells compared with CD8+ cells. Grade ≥3 cytokine release syndrome was associated with polyfunctional T cells, and both grade ≥3 neurologic toxicity and antitumor efficacy were associated with polyfunctional IL-17A-producing T cells. The findings in this exploratory study show that a preinfusion CAR product T-cell subset with a definable polyfunctional profile has a major association with clinical outcomes of CAR T-cell therapy. This trial was registered at www.clinicaltrials.gov as #NCT00924326.

A centromere satellite concomitant with extensive karyotypic diversity across the Peromyscus genus defies predictions of molecular drive.

A common feature of eukaryotic centromeres is the presence of large tracts of tandemly arranged repeats, known as satellite DNA. However, these centromeric repeats appear to experience rapid evolution under forces such as molecular drive and centromere drive, seemingly without consequence to the integrity of the centromere. Moreover, blocks of heterochromatin within the karyotype, including the centromere, are hotspots for chromosome rearrangements that may drive speciation events by contributing to reproductive isolation. However, the relationship between the evolution of heterochromatic sequences and the karyotypic dynamics of these regions remains largely unknown. Here, we show that a single conserved satellite DNA sequence in the order Rodentia of the genus Peromyscus localizes to recurrent sites of chromosome rearrangements and heterochromatic amplifications. Peromyscine species display several unique features of chromosome evolution compared to other Rodentia, including stable maintenance of a strict chromosome number of 48 among all known species in the absence of any detectable interchromosomal rearrangements. Rather, the diverse karyotypes of Peromyscine species are due to intrachromosomal variation in blocks of repeated DNA content. Despite wide variation in the copy number and location of repeat blocks among different species, we find that a single satellite monomer maintains a conserved sequence and homogenized tandem repeat structure, defying predictions of molecular drive. The conservation of this satellite monomer results in common, abundant, and large blocks of chromatin that are homologous among chromosomes within one species and among diverged species. Thus, such a conserved repeat may have facilitated the retention of polymorphic chromosome variants within individuals and intrachromosomal rearrangements between species-both factors that have previously been hypothesized to contribute towards the extremely wide range of ecological adaptations that this genus exhibits.

Reshaping the Tumor Microenvironment of KRASG12D Pancreatic Ductal Adenocarcinoma with Combined SOS1 and MEK Inhibition for Improved Immunotherapy Response.

KRAS inhibitors have demonstrated exciting preclinical and clinical responses, although resistance occurs rapidly. Here, we investigate the effects of KRAS-targeting therapies on the tumor microenvironment using a library of KrasG12D, p53-mutant, murine pancreatic ductal adenocarcinoma-derived cell lines (KPCY) to leverage immune-oncology combination strategies for long-term tumor efficacy. Our findings show that SOS1 and MEK inhibitors (SOS1i+MEKi) suppressed tumor growth in syngeneic models and increased intratumoral CD8+ T cells without durable responses. Single-cell RNA sequencing revealed an increase in inflammatory cancer-associated fibroblasts (iCAF), M2 macrophages, and a decreased dendritic cell (DC) quality that ultimately resulted in a highly immunosuppressive microenvironment driven by IL6+ iCAFs. Agonist CD40 treatment was effective to revert macrophage polarization and overcome the lack of mature antigen-presenting DCs after SOS1i+MEKi therapy. Treatment increased the overall survival of KPCY tumor-bearing mice. The addition of checkpoint blockade to SOS1i+MEKi combination resulted in tumor-free mice with established immune memory. Our data suggest that KRAS inhibition affects myeloid cell maturation and highlights the need for combining KRAS cancer-targeted therapy with myeloid activation to enhance and prolong antitumor effects. SIGNIFICANCE: Combination of SOS1 and MEK inhibitors increase T cell infiltration while blunting pro-immune myeloid cell maturation and highlights the need for combining KRAS cancer-targeted therapy with myeloid activation to enhance and prolong anti-tumor effects.

The genetic architecture of the human skeletal form.

The human skeletal form underlies our ability to walk on two legs, but unlike standing height, the genetic basis of limb lengths and skeletal proportions is less well understood. Here we applied a deep learning model to 31,221 whole body dual-energy X-ray absorptiometry (DXA) images from the UK Biobank (UKB) to extract 23 different image-derived phenotypes (IDPs) that include all long bone lengths as well as hip and shoulder width, which we analyzed while controlling for height. All skeletal proportions are highly heritable (∻40-50%), and genome-wide association studies (GWAS) of these traits identified 179 independent loci, of which 102 loci were not associated with height. These loci are enriched in genes regulating skeletal development as well as associated with rare human skeletal diseases and abnormal mouse skeletal phenotypes. Genetic correlation and genomic structural equation modeling indicated that limb proportions exhibited strong genetic sharing but were genetically independent of width and torso proportions. Phenotypic and polygenic risk score analyses identified specific associations between osteoarthritis (OA) of the hip and knee, the leading causes of adult disability in the United States, and skeletal proportions of the corresponding regions. We also found genomic evidence of evolutionary change in arm-to-leg and hip-width proportions in humans consistent with striking anatomical changes in these skeletal proportions in the hominin fossil record. In contrast to cardiovascular, auto-immune, metabolic, and other categories of traits, loci associated with these skeletal proportions are significantly enriched in human accelerated regions (HARs), and regulatory elements of genes differentially expressed through development between humans and the great apes. Taken together, our work validates the use of deep learning models on DXA images to identify novel and specific genetic variants affecting the human skeletal form and ties a major evolutionary facet of human anatomical change to pathogenesis.

The genetic architecture and evolution of the human skeletal form.

The human skeletal form underlies bipedalism, but the genetic basis of skeletal proportions (SPs) is not well characterized. We applied deep-learning models to 31,221 x-rays from the UK Biobank to extract a comprehensive set of SPs, which were associated with 145 independent loci genome-wide. Structural equation modeling suggested that limb proportions exhibited strong genetic sharing but were independent of width and torso proportions. Polygenic score analysis identified specific associations between osteoarthritis and hip and knee SPs. In contrast to other traits, SP loci were enriched in human accelerated regions and in regulatory elements of genes that are differentially expressed between humans and great apes. Combined, our work identifies specific genetic variants that affect the skeletal form and ties a major evolutionary facet of human anatomical change to pathogenesis.

Deep learning based phenotyping of medical images improves power for gene discovery of complex disease.

Electronic health records are often incomplete, reducing the power of genetic association studies. For some diseases, such as knee osteoarthritis where the routine course of diagnosis involves an X-ray, image-based phenotyping offers an alternate and unbiased way to ascertain disease cases. We investigated this by training a deep-learning model to ascertain knee osteoarthritis cases from knee DXA scans that achieved clinician-level performance. Using our model, we identified 1931 (178%) more cases than currently diagnosed in the health record. Individuals diagnosed as cases by our model had higher rates of self-reported knee pain, for longer durations and with increased severity compared to control individuals. We trained another deep-learning model to measure the knee joint space width, a quantitative phenotype linked to knee osteoarthritis severity. In performing genetic association analysis, we found that use of a quantitative measure improved the number of genome-wide significant loci we discovered by an order of magnitude compared with our binary model of cases and controls despite the two phenotypes being highly genetically correlated. In addition we discovered associations between our quantitative measure of knee osteoarthritis and increased risk of adult fractures- a leading cause of injury-related death in older individuals-, illustrating the capability of image-based phenotyping to reveal epidemiological associations not captured in the electronic health record. For diseases with radiographic diagnosis, our results demonstrate the potential for using deep learning to phenotype at biobank scale, improving power for both genetic and epidemiological association analysis.

Effects of localized neurotrophin gene expression on spiral ganglion neuron resprouting in the deafened cochlea.

A cochlear implant may be used to electrically stimulate spiral ganglion neurons (SGNs) in people with severe sensorineural hearing loss (SNHL). However, these neurons progressively degenerate after SNHL due to loss of neurotrophins normally supplied by sensory hair cells (HCs). Experimentally, exogenous neurotrophin administration prevents SGN degeneration but can also result in abnormal resprouting of their peripheral fibers. This study aimed to create a target-derived neurotrophin source to increase neuron survival and redirect fiber resprouting following SNHL. Adenoviral (Ad) vectors expressing green fluorescent protein (GFP) alone or in combination with brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT3) were injected into the cochlear scala tympani or scala media of guinea-pigs (GPs) deafened via aminoglycosides for 1 week. After 3 weeks, cochleae were examined for gene expression, neuron survival, and the projection of peripheral fibers in response to gene expression. Injection of vectors into the scala media resulted in more localized gene expression than scala tympani injection with gene expression consistently observed within the partially degenerated organ of Corti. There was also greater neuron survival and evidence of localized fiber responses to neurotrophin-expressing cells within the organ of Corti from scala media injections (P < 0.05), a first step in promoting organized resprouting of auditory peripheral fibers via gene therapy.

A radiolabeled drug tracing method to study neurotrophin-3 retention and distribution in the cochlea after nano-based local delivery.

Hearing loss is the most common sensory deficit worldwide with no approved therapeutics for treatment. Local neurotrophin delivery into the cochlea has shown great potential in protecting and repairing the sensory cells important for hearing. However, delivery of these factors into the inner ear at therapeutic levels over a sustained period of time has remained a challenge restricting clinical translation. We have developed a method to test the pharmacokinetics of neurotrophin released from porous silica particles called 'supraparticles' that can provide sustained release of neurotrophins to the inner ear.•This report describes a radiolabeling method to examine neurotrophin retention and distribution in the cochlea. The neurotrophin was labeled with a radioactive tracer (iodine 125: 125I) and delivered into the cochlea via the supraparticle system.•Gamma counts reveal drug levels and clearance in the intact cochlea, as well as accumulation in off-target organs (safety test). Autoradiography analyses using film and emulsion permit quantification and visualization of drug distribution at the cellular level. The method has a detection limit of 0.8 pg of radiolabeled neurotrophin-3 in cochlear sections exposed to film.•The tracer 125I with a half-life of 59.4 days can be used to label other drugs/substances with a tyrosine residue and therefore be broadly applicable for long-term pharmacokinetic studies in other systems.

Chronic intracochlear electrical stimulation at high charge densities: reducing platinum dissolution.

OBJECTIVE: Cochleae of long-term cochlear implant users have shown evidence of particulate platinum (Pt) corroded from the surface of Pt electrodes. The pathophysiological effect of Pt within the cochlea has not been extensively investigated. We previously evaluated the effects of Pt corrosion at high charge densities and reported negligible pathophysiological impact. The present study extends this work by examining techniques that may reduce Pt corrosion. APPROACH: Deafened guinea pigs were continuously stimulated for 28 d using biphasic current pulses at extreme charge densities using: (i) electrode shorting; (ii) electrode shorting with capacitive coupling (CC); or (iii) electrode shorting with alternating leading phase (AP). On completion of stimulation, cochleae were examined for corrosion product, tissue response, auditory nerve (AN) survival and trace levels of Pt; and electrodes examined for surface corrosion. MAIN RESULTS: Pt corrosion was evident at ≥200 µC cm-2 phase-1; the amount dependent on charge density (p< 0.01) and charge recovery technique (p < 0.01); reduced corrosion was apparent using CC. Tissue response increased with charge density (p< 0.007); cochleae stimulated at ≥200 µC cm-2 phase-1 exhibited a vigorous response including a focal region of necrosis and macrophages. Notably, tissue response was not dependent on the charge recovery technique (p = 0.56). Despite stimulation at high charge densities resulting in significant levels of Pt corrosion, there was no stimulus induced loss of ANs. SIGNIFICANCE: Significant increases in tissue response and Pt corrosion were observed following stimulation at high charge densities. Charge recovery using CC, and to a lesser extent AP, reduced the amount of Pt corrosion but not the tissue response. Stimulation at change densities an order of magnitude higher than those used when programming cochlear implant recipients in the clinic, produced a vigorous tissue response and corrosion products without evidence of neural loss.

Pharmacokinetics and tissue distribution of neurotrophin 3 after intracochlear delivery.

Neurotrophin therapy has potential to reverse some forms of hearing loss. However, cochlear pharmacokinetic studies are challenging due to small fluid volumes. Here a radioactive tracer was used to determine neurotrophin-3 retention, distribution and clearance after intracochlear administration. 125I-neurotrophin-3 was injected into guinea pig cochleae using a sealed injection technique comparing dosing volumes, rates and concentrations up to 750 µg/mL. Retention was measured by whole-cochlear gamma counts at five time points while distribution and clearance were assessed by autoradiography. Smaller injection volumes and higher concentrations correlated with higher retention of neurotrophin-3. Distribution of neurotrophin-3 was widespread throughout the cochlear tissue, decreasing in concentration from base to apex. Tissue distribution was non-uniform, with greatest density in cells lining the scala tympani and lower density in neural target tissue. The time constant for clearance of neurotrophin-3 from cochlear tissues was 38 h but neurotrophin-3 remained detectable for at least 2 weeks. Neurotrophin-3 was evident in the semi-circular canals with minor spread to the contralateral cochlea. This study is the first comprehensive evaluation of the disposition profile for a protein therapy in the cochlea. The findings and methods in this study will provide valuable guidance for the development of protein therapies for hearing loss.

Hair cell regeneration after ATOH1 gene therapy in the cochlea of profoundly deaf adult guinea pigs.

The degeneration of hair cells in the mammalian cochlea results in permanent sensorineural hearing loss. This study aimed to promote the regeneration of sensory hair cells in the mature cochlea and their reconnection with auditory neurons through the introduction of ATOH1, a transcription factor known to be necessary for hair cell development, and the introduction of neurotrophic factors. Adenoviral vectors containing ATOH1 alone, or with neurotrophin-3 and brain derived neurotrophic factor were injected into the lower basal scala media of guinea pig cochleae four days post ototoxic deafening. Guinea pigs treated with ATOH1 gene therapy, alone, had a significantly greater number of cells expressing hair cell markers compared to the contralateral non-treated cochlea when examined 3 weeks post-treatment. This increase, however, did not result in a commensurate improvement in hearing thresholds, nor was there an increase in synaptic ribbons, as measured by CtBP2 puncta after ATOH1 treatment alone, or when combined with neurotrophins. However, hair cell formation and synaptogenesis after co-treatment with ATOH1 and neurotrophic factors remain inconclusive as viral transduction was reduced due to the halving of viral titres when the samples were combined. Collectively, these data suggest that, whilst ATOH1 alone can drive non-sensory cells towards an immature sensory hair cell phenotype in the mature cochlea, this does not result in functional improvements after aminoglycoside-induced deafness.

Viability of long-term gene therapy in the cochlea.

Gene therapy has been investigated as a way to introduce a variety of genes to treat neurological disorders. An important clinical consideration is its long-term effectiveness. This research aims to study the long-term expression and effectiveness of gene therapy in promoting spiral ganglion neuron survival after deafness. Adenoviral vectors modified to express brain derived neurotrophic factor or neurotrophin-3 were unilaterally injected into the guinea pig cochlea one week post ototoxic deafening. After six months, persistence of gene expression and significantly greater neuronal survival in neurotrophin-treated cochleae compared to the contralateral cochleae were observed. The long-term gene expression observed indicates that gene therapy is potentially viable; however the degeneration of the transduced cells as a result of the original ototoxic insult may limit clinical effectiveness. With further research aimed at transducing stable cochlear cells, gene therapy may be an efficacious way to introduce neurotrophins to promote neuronal survival after hearing loss.

Neurotrophin gene therapy for sustained neural preservation after deafness.

The cochlear implant provides auditory cues to profoundly deaf patients by electrically stimulating the residual spiral ganglion neurons. These neurons, however, undergo progressive degeneration after hearing loss, marked initially by peripheral fibre retraction and ultimately culminating in cell death. This research aims to use gene therapy techniques to both hold and reverse this degeneration by providing a sustained and localised source of neurotrophins to the deafened cochlea. Adenoviral vectors containing green fluorescent protein, with or without neurotrophin-3 and brain derived neurotrophic factor, were injected into the lower basal turn of scala media of guinea pigs ototoxically deafened one week prior to intervention. This single injection resulted in localised and sustained gene expression, principally in the supporting cells within the organ of Corti. Guinea pigs treated with adenoviral neurotrophin-gene therapy had greater neuronal survival compared to contralateral non-treated cochleae when examined at 7 and 11 weeks post injection. Moreover; there was evidence of directed peripheral fibre regrowth towards cells expressing neurotrophin genes after both treatment periods. These data suggest that neurotrophin-gene therapy can provide sustained protection of spiral ganglion neurons and peripheral fibres after hearing loss.

The effect of deafness duration on neurotrophin gene therapy for spiral ganglion neuron protection.

A cochlear implant can restore hearing function by electrically exciting spiral ganglion neurons (SGNs) in the deaf cochlea. However, following deafness SGNs undergo progressive degeneration ultimately leading to their death. One significant cause of SGN degeneration is the loss of neurotrophic support that is normally provided by cells within the organ of Corti (OC). The administration of exogenous neurotrophins (NTs) can protect SGNs from degeneration but the effects are short-lived once the source of NTs has been exhausted. NT gene therapy, whereby cells within the cochlea are transfected with genes enabling them to produce NTs, is one strategy for providing a cellular source of NTs that may provide long-term support for SGNs. As the SGNs normally innervate sensory cells within the OC, targeting residual OC cells for gene therapy in the deaf cochlea may provide a source of NTs for SGN protection and targeted regrowth of their peripheral fibers. However, the continual degeneration of the OC over extended periods of deafness may deplete the cellular targets for NT gene therapy and hence limit the effectiveness of this method in preventing SGN loss. This study examined the effects of deafness duration on the efficacy of NT gene therapy in preventing SGN loss in guinea pigs that were systemically deafened with aminoglycosides. Adenoviral vectors containing green fluorescent protein (GFP) with or without genes for Brain Derived Neurotrophic Factor (BDNF) and Neurotrophin-3 (NT3) were injected into the scala media (SM) compartment of cochleae that had been deafened for one, four or eight weeks prior to the viral injection. The results showed that viral transfection of cells within the SM was still possible even after severe degeneration of the OC. Supporting cells (pillar and Deiters' cells), cells within the stria vascularis, the spiral ligament, endosteal cells lining the scala compartments and interdental cells in the spiral limbus were transfected. However, the level of transfection was remarkably lower following longer durations of deafness. There was a significant increase in SGN survival in the entire basal turn for cochleae that received NT gene therapy compared to the untreated contralateral control cochleae for the one week deaf group. In the four week deaf group significant SGN survival was observed in the lower basal turn only. There was no increase in SGN survival for the eight week deaf group in any cochlear region. These findings indicated that the efficacy of NT gene therapy diminished with increasing durations of deafness leading to reduced benefits in terms of SGN protection. Clinically, there remains a window of opportunity in which NT gene therapy can provide ongoing trophic support for SGNs.

Polypyrrole-coated electrodes for the delivery of charge and neurotrophins to cochlear neurons.

Sensorineural hearing loss is associated with gradual degeneration of spiral ganglion neurons (SGNs), compromising hearing outcomes with cochlear implant use. Combination of neurotrophin delivery to the cochlea and electrical stimulation from a cochlear implant protects SGNs, prompting research into neurotrophin-eluting polymer electrode coatings. The electrically conducting polypyrrole/para-toluene sulfonate containing neurotrophin-3 (Ppy/pTS/NT3) was applied to 1.7 mm2 cochlear implant electrodes. Ppy/pTS/NT3-coated electrode arrays stored 2 ng NT3 and released 0.1 ng/day with electrical stimulation. Guinea pigs were implanted with Ppy/pTS or Ppy/pTS/NT3 electrode arrays two weeks after deafening via aminoglycosides. The electrodes of a subgroup of these guinea pigs were electrically stimulated for 8 h/day for 2 weeks. There was a loss of SGNs in the implanted cochleae of guinea pigs with Ppy/pTS-coated electrodes indicative of electrode insertion damage. However, guinea pigs implanted with electrically stimulated Ppy/pTS/NT3-coated electrodes had lower electrically-evoked auditory brainstem response thresholds and greater SGN densities in implanted cochleae compared to non-implanted cochleae and compared to animals implanted with Ppy/pTS-coated electrodes (p<0.05). Ppy/pTS/NT3 did not exacerbate fibrous tissue formation and did not affect electrode impedance. Drug-eluting conducting polymer coatings on cochlear implant electrodes present a clinically viable method to promote preservation of SGNs without adversely affecting the function of the cochlear implant.

Effect of Gdnf haploinsufficiency on rate of migration and number of enteric neural crest-derived cells.

The enteric nervous system arises predominantly from vagal level neural crest cells that migrate into the foregut and then colonize the entire length of the gastrointestinal tract. Previous studies have demonstrated that glial cell line-derived neurotrophic factor (GDNF) promotes the migration of enteric neural crest-derived cells (ENCs) in vitro, but a role for GDNF in the migration of ENCs in vivo has yet to be demonstrated. In this study, the effects of Gdnf haploinsufficiency on ENC rate of migration and number during mid embryonic development were examined. Although the entire gut of embryonic Gdnf(+/-) mice was colonized, a significant delay in the migration of ENCs along the embryonic hindgut was found. However, significant effects of Gdnf haploinsufficiency on ENC number were detected before the stage at which migration defects were first evident. As previous studies have shown a relationship between ENC number and migration, the effects of Gdnf haploinsufficiency on migration may be due to an indirect effect on cell number and/or a direct effect of GDNF on ENC migration. Gdnf haploinsufficiency did not cause any detectable change in the rate of neuronal differentiation of ENCs.