Engineering T cell receptor fusion proteins using nonviral CRISPR/Cas9 genome editing for cancer immunotherapy.

Manufacture of chimeric antigen receptor (CAR)-T cells usually involves the use of viral delivery systems to achieve high transgene expression. However, it can be costly and may result in random integration of the CAR into the genome, creating several disadvantages including variation in transgene expression, functional gene silencing and potential oncogenic transformation. Here, we optimized the method of nonviral, CRISPR/Cas9 genome editing using large donor DNA delivery, knocked-in an anti-tumor single chain variable fragment (scFv) into the N-terminus of CD3ε and efficiently generated fusion protein (FP) T cells. These cells displayed FP integration within the TCR/CD3 complex, lower variability in gene expression compared to CAR-T cells and good cell expansion after transfection. CD3ε FP T cells were predominantly CD8+ effector memory T cells, and exhibited anti-tumor activity in vitro and in vivo. Dual targeting FP T cells were also generated through the incorporation of scFvs into other CD3 subunits and CD28. Compared to viral-based methods, this method serves as an alternative and versatile way of generating T cells with tumor-targeting receptors for cancer immunotherapy.

Mitoribosome sensitivity to HSP70 inhibition uncovers metabolic liabilities of castration-resistant prostate cancer.

The androgen receptor is a key regulator of prostate cancer and the principal target of current prostate cancer therapies collectively termed androgen deprivation therapies. Insensitivity to these drugs is a hallmark of progression to a terminal disease state termed castration-resistant prostate cancer. Therefore, novel therapeutic options that slow progression of castration-resistant prostate cancer and combine effectively with existing agents are in urgent need. We show that JG-98, an allosteric inhibitor of HSP70, re-sensitizes castration-resistant prostate cancer to androgen deprivation drugs by targeting mitochondrial HSP70 (HSPA9) to suppress aerobic respiration. Rather than impacting androgen receptor stability as previously described, JG-98's primary effect is inhibition of mitochondrial translation, leading to disruption of electron transport chain activity. Although functionally distinct from HSPA9 inhibition, direct inhibition of the electron transport chain with a complex I or II inhibitor creates a similar physiological state capable of re-sensitizing castration-resistant prostate cancer to androgen deprivation therapies. These data identify a significant role for HspA9 in mitochondrial ribosome function and highlight an actionable metabolic vulnerability of castration-resistant prostate cancer.

The rate of telomere loss is related to maximum lifespan in birds.

Telomeres are highly conserved regions of DNA that protect the ends of linear chromosomes. The loss of telomeres can signal an irreversible change to a cell's state, including cellular senescence. Senescent cells no longer divide and can damage nearby healthy cells, thus potentially placing them at the crossroads of cancer and ageing. While the epidemiology, cellular and molecular biology of telomeres are well studied, a newer field exploring telomere biology in the context of ecology and evolution is just emerging. With work to date focusing on how telomere shortening relates to individual mortality, less is known about how telomeres relate to ageing rates across species. Here, we investigated telomere length in cross-sectional samples from 19 bird species to determine how rates of telomere loss relate to interspecific variation in maximum lifespan. We found that bird species with longer lifespans lose fewer telomeric repeats each year compared with species with shorter lifespans. In addition, phylogenetic analysis revealed that the rate of telomere loss is evolutionarily conserved within bird families. This suggests that the physiological causes of telomere shortening, or the ability to maintain telomeres, are features that may be responsible for, or co-evolved with, different lifespans observed across species.This article is part of the theme issue 'Understanding diversity in telomere dynamics'.

Telomerase activity is maintained throughout the lifespan of long-lived birds.

Telomerase is an enzyme capable of elongating telomeres, the caps at the ends of chromosomes associated with aging, lifespan and survival. We investigated tissue-level variation in telomerase across different ages in four bird species that vary widely in their life history. Telomerase activity in bone marrow may be associated with the rate of erythrocyte telomere shortening; birds with lower rates of telomere shortening and longer lifespans have higher bone marrow telomerase activity throughout life. Telomerase activity in all of the species appears to be tightly correlated with the proliferative potential of specific organs, and it is also highest in the hatchling age-class, when the proliferative demands of most organs are the highest. This study offers an alternative view to the commonly held hypothesis that telomerase activity is down-regulated in all post-mitotic somatic tissues in long-lived organisms as a tumor-protective mechanism. This highlights the need for more comparative analyses of telomerase, lifespan and the incidence of tumor formation.

Telomerase expression is differentially regulated in birds of differing life span.

Cellular senescence caused by telomere shortening has been suggested as one potential causal agent of aging. In some tissues, telomeres are maintained by telomerase; however, telomerase promotes tumor formation, suggesting a trade-off between aging and cancer. We predicted that telomerase activity should vary directly with life span. We determined telomerase activity in bone marrow in cross-sectional samples from two short-lived bird species and two long-lived bird species. The two short-lived species had high telomerase activity as hatchlings but showed a sharp downregulation in both the young and old adults, whereas the two long-lived species had relatively high telomerase activity in bone marrow that did not decrease with age. In zebra finches, the age-related change in telomerase activity varied in different tissues. Telomerase activity increased late in life in skeletal muscle, liver, and gonad, but not in blood or bone marrow.