The antifibrotic drug pirfenidone promotes pulmonary cavitation and drug resistance in a mouse model of chronic tuberculosis

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Abstract

Pirfenidone is a recently approved antifibrotic drug for the treatment of idiopathic pulmonary fibrosis (IPF). Because tuberculosis (TB) is characterized by granulomatous inflammation in conjunction with parenchymal destruction and replacement fibrosis, we sought to determine whether the addition of pirfenidone as an adjunctive, host-directed therapy provides a beneficial effect during antimicrobial treatment of TB. We hypothesized that pirfenidone’s antiinflammatory and antifibrotic properties would reduce inflammatory lung damage and increase antimicrobial drug penetration in granulomas to accelerate treatment response. The effectiveness of adjunctive pirfenidone during TB drug therapy was evaluated using a murine model of chronic TB. Mice treated with standard therapy 2HRZ/4HR (H, isoniazid; R, rifampin; and Z, pyrazinamide) were compared with 2 alternative regimens containing pirfenidone (Pf) (2HRZPf/4HRPf and 2HRZPf/4HR). Contrary to our hypothesis, adjunctive pirfenidone use leads to reduced bacterial clearance and increased relapse rates. This treatment failure is closely associated with the emergence of isoniazid monoresistant bacilli, increased cavitation, and significant lung pathology. While antifibrotic agents may eventually be used as part of adjunctive host-directed therapy of TB, this study clearly demonstrates that caution must be exercised. Moreover, as pirfenidone becomes more widely used in clinical practice, increased patient monitoring would be required in endemic TB settings.

Authors

Bintou A. Ahidjo, Mariama C. Maiga, Elizabeth A. Ihms, Mamoudou Maiga, Alvaro A. Ordonez, Laurene S. Cheung, Sarah Beck, Bruno B. Andrade, Sanjay Jain, William R. Bishai

T cell responses to human platelet antigen–1a involve a unique form of indirect allorecognition

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Abstract

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a pregnancy-related condition caused by maternal antibodies binding an alloantigen on fetal platelets. In most cases the alloantigen is formed by a single amino acid, integrin β3 Leu33, referred to as human platelet antigen–1a (HPA-1a). Production of anti–HPA-1a antibodies likely depends on CD4⁺ T cells that recognize the same alloantigen in complex with the HLA-DRA/DRB3*01:01 molecule. While this complex is well characterized, T cell recognition of it is not. Here, to examine the nature of antigen recognition by HPA-1a–specific T cells, we assayed native and synthetic variants of the integrin β3 peptide antigen for binding to DRA/DRB3*01:01-positive antigen-presenting cells and for T cell activation. We found that HPA-1a–specific T cells recognize non-allogeneic integrin β3 residues anchored to DRA/DRB3*01:01 by the allogeneic Leu33, which itself is not directly recognized by these T cells. Furthermore, these T cell responses are diverse, with different T cells depending on different residues for recognition. This represents a unique form of indirect allorecognition in which a non-allogeneic peptide sequence becomes immunogenic by stable anchoring to MHC by an allogeneic residue.

Authors

Maria Therese Ahlen, Anne Husebekk, Ida Løken Killie, Bjørn Skogen, Tor Brynjar Stuge

Telomere dysfunction in alveolar epithelial cells causes lung remodeling and fibrosis

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Abstract

Telomeres are short in type II alveolar epithelial cells (AECs) of patients with idiopathic pulmonary fibrosis (IPF). Whether dysfunctional telomeres contribute directly to development of lung fibrosis remains unknown. The objective of this study was to investigate whether telomere dysfunction in type II AECs, mediated by deletion of the telomere shelterin protein TRF1, leads to pulmonary fibrosis in mice (SPC-Cre TRF1^fl/fl mice). Deletion of TRF1 in type II AECs for 2 weeks increased γH2AX DNA damage foci, but not histopathologic changes in the lung. Deletion of TRF1 in type II AECs for up to 9 months resulted in short telomeres and lung remodeling characterized by increased numbers of type II AECs, α-smooth muscle actin⁺ mesenchymal cells, collagen deposition, and accumulation of senescence-associated β-galactosidase⁺ lung epithelial cells. Deletion of TRF1 in collagen-expressing cells caused pulmonary edema, but not fibrosis. These results demonstrate that prolonged telomere dysfunction in type II AECs, but not collagen-expressing cells, leads to age-dependent lung remodeling and fibrosis. We conclude that telomere dysfunction in type II AECs is sufficient to cause lung fibrosis, and may be a dominant molecular defect causing IPF. SPC-Cre TRF1^fl/fl mice will be useful for assessing cellular and molecular mechanisms of lung fibrosis mediated by telomere dysfunction.

Authors

Ram P. Naikawadi, Supparerk Disayabutr, Benat Mallavia, Matthew L. Donne, Gary Green, Janet L. La, Jason R. Rock, Mark R. Looney, Paul J. Wolters

IL1RL1 asthma risk variants regulate airway type 2 inflammation

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Abstract

Genome-wide association studies of asthma have identified genetic variants in the IL1RL1 gene, but the molecular mechanisms conferring risk are unknown. IL1RL1 encodes the ST2 receptor (ST2L) for IL-33 and an inhibitory decoy receptor (sST2). IL-33 promotes type 2 inflammation, which is present in some but not all asthmatics. We find that two single nucleotide polymorphisms (SNPs) in IL1RL1 — rs1420101 and rs11685480 — are strongly associated with plasma sST2 levels, though neither is an expression quantitative trait locus (eQTL) in whole blood. Rather, rs1420101 and rs11685480 mark eQTLs in airway epithelial cells and distal lung parenchyma, respectively. We find that the genetically determined plasma sST2 reservoir, derived from the lung, neutralizes IL-33 activity, and these eQTL SNPs additively increase the risk of airway type 2 inflammation among asthmatics. These risk variants define a population of asthmatics at risk of IL-33–driven type 2 inflammation.

Authors

Erin D. Gordon, Joe Palandra, Agata Wesolowska-Andersen, Lando Ringel, Cydney L. Rios, Marrah E. Lachowicz-Scroggins, Louis Z. Sharp, Jamie L. Everman, Hannah J. MacLeod, Jae W. Lee, Robert J. Mason, Michael A. Matthay, Richard T. Sheldon, Michael C. Peters, Karl H. Nocka, John V. Fahy, Max A. Seibold

Glymphatic fluid transport controls paravascular clearance of AAV vectors from the brain

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Abstract

Adeno-associated viruses (AAV) are currently being evaluated in clinical trials for gene therapy of CNS disorders. However, host factors that influence the spread, clearance, and transduction efficiency of AAV vectors in the brain are not well understood. Recent studies have demonstrated that fluid flow mediated by aquaporin-4 (AQP4) channels located on astroglial end feet is essential for exchange of solutes between interstitial and cerebrospinal fluid. This phenomenon, which is essential for interstitial clearance of solutes from the CNS, has been termed glial-associated lymphatic transport or glymphatic transport. In the current study, we demonstrate that glymphatic transport profoundly affects various aspects of AAV gene transfer in the CNS. Altered localization of AQP4 in aged mouse brains correlated with significantly increased retention of AAV vectors in the parenchyma and reduced systemic leakage following ventricular administration. We observed a similar increase in AAV retention and transgene expression upon i.c.v. administration in AQP4^–/– mice. Consistent with this observation, fluorophore-labeled AAV vectors showed markedly reduced flux from the ventricles of AQP4^–/– mice compared with WT mice. These results were further corroborated by reduced AAV clearance from the AQP4-null brain, as demonstrated by reduced transgene expression and vector genome accumulation in systemic organs. We postulate that deregulation of glymphatic transport in aged and diseased brains could markedly affect the parenchymal spread, clearance, and gene transfer efficiency of AAV vectors. Assessment of biomarkers that report the kinetics of CSF flux in prospective gene therapy patients might inform variable treatment outcomes and guide future clinical trial design.

Authors

Giridhar Murlidharan, Andrew Crowther, Rebecca A. Reardon, Juan Song, Aravind Asokan

Platelet integrin α6β1 controls lung metastasis through direct binding to cancer cell–derived ADAM9

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Abstract

Metastatic dissemination of cancer cells, which accounts for 90% of cancer mortality, is the ultimate hallmark of malignancy. Growing evidence suggests that blood platelets have a predominant role in tumor metastasis; however, the molecular mechanisms involved remain elusive. Here, we demonstrate that genetic deficiency of integrin α6β1 on platelets markedly decreases experimental and spontaneous lung metastasis. In vitro and in vivo assays reveal that human and mouse platelet α6β1 supports platelet adhesion to various types of cancer cells. Using a knockdown approach, we identified ADAM9 as the major counter receptor of α6β1 on both human and mouse tumor cells. Static and flow-based adhesion assays of platelets binding to DC-9, a recombinant protein covering the disintegrin-cysteine domain of ADAM9, demonstrated that this receptor directly binds to platelet α6β1. In vivo studies showed that the interplay between platelet α6β1 and tumor cell–expressed ADAM9 promotes efficient lung metastasis. The integrin α6β1–dependent platelet-tumor cell interaction induces platelet activation and favors the extravasation process of tumor cells. Finally, we demonstrate that a pharmacological approach targeting α6β1 efficiently impairs tumor metastasis through a platelet-dependent mechanism. Our study reveals a mechanism by which platelets promote tumor metastasis and suggests that integrin α6β1 represents a promising target for antimetastatic therapies.

Authors

Elmina Mammadova-Bach, Paola Zigrino, Camille Brucker, Catherine Bourdon, Monique Freund, Adèle De Arcangelis, Scott I. Abrams, Gertaud Orend, Christian Gachet, Pierre Henri Mangin

Lack of immunoediting in murine pancreatic cancer reversed with neoantigen

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Abstract

In carcinogen-driven cancers, a high mutational burden results in neoepitopes that can be recognized immunologically. Such carcinogen-induced tumors may evade this immune response through “immunoediting,” whereby tumors adapt to immune pressure and escape T cell–mediated killing. Many tumors lack a high neoepitope burden, and it remains unclear whether immunoediting occurs in such cases. Here, we evaluated T cell immunity in an autochthonous mouse model of pancreatic cancer and found a low mutational burden, absence of predicted neoepitopes derived from tumor mutations, and resistance to checkpoint immunotherapy. Spontaneous tumor progression was identical in the presence or absence of T cells. Moreover, tumors arising in T cell–depleted mice grew unchecked in immune-competent hosts. However, introduction of the neoantigen ovalbumin (OVA) led to tumor rejection and T cell memory, but this did not occur in OVA immune-tolerant mice. Thus, immunoediting does not occur in this mouse model — a likely consequence, not a cause, of absent neoepitopes. Because many human tumors also have a low missense mutational load and minimal neoepitope burden, our findings have clinical implications for the design of immunotherapy for patients with such tumors.

Authors

Rebecca A. Evans, Mark S. Diamond, Andrew J. Rech, Timothy Chao, Max W. Richardson, Jeffrey H. Lin, David L. Bajor, Katelyn T. Byrne, Ben Z. Stanger, James L. Riley, Nune Markosyan, Rafael Winograd, Robert H. Vonderheide

In vivo disruption of latent HSV by designer endonuclease therapy

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Abstract

A large portion of the global population carries latent herpes simplex virus (HSV), which can periodically reactivate, resulting in asymptomatic shedding or formation of ulcerative lesions. Current anti-HSV drugs do not eliminate latent virus from sensory neurons where HSV resides, and therefore do not eliminate the risk of transmission or recurrent disease. Here, we report the ability of HSV-specific endonucleases to induce mutations of essential HSV genes both in cultured neurons and in latently infected mice. In neurons, viral genomes are susceptible to endonuclease-mediated mutagenesis, regardless of the time of treatment after HSV infection, suggesting that both HSV lytic and latent forms can be targeted. Mutagenesis frequency after endonuclease exposure can be increased nearly 2-fold by treatment with a histone deacetylase (HDAC) inhibitor. Using a mouse model of latent HSV infection, we demonstrate that a targeted endonuclease can be delivered to viral latency sites via an adeno-associated virus (AAV) vector, where it is able to induce mutation of latent HSV genomes. These data provide the first proof-of-principle to our knowledge for the use of a targeted endonuclease as an antiviral agent to treat an established latent viral infection in vivo.

Authors

Martine Aubert, Emily A. Madden, Michelle Loprieno, Harshana S. DeSilva Feelixge, Laurence Stensland, Meei-Li Huang, Alexander L. Greninger, Pavitra Roychoudhury, Nixon Niyonzima, Thuy Nguyen, Amalia Magaret, Roman Galleto, Daniel Stone, Keith R. Jerome

CFTR gene transfer with AAV improves early cystic fibrosis pig phenotypes

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Abstract

The physiological components that contribute to cystic fibrosis (CF) lung disease are steadily being elucidated. Gene therapy could potentially correct these defects. CFTR-null pigs provide a relevant model to test gene therapy vectors. Using an in vivo selection strategy that amplifies successful capsids by replicating their genomes with helper adenovirus coinfection, we selected an adeno-associated virus (AAV) with tropism for pig airway epithelia. The evolved capsid, termed AAV2H22, is based on AAV2 with 5 point mutations that result in a 240-fold increased infection efficiency. In contrast to AAV2, AAV2H22 binds specifically to pig airway epithelia and is less reliant on heparan sulfate for transduction. We administer AAV2H22-CFTR expressing the CF transmembrane conductance regulator (CFTR) cDNA to the airways of CF pigs. The transduced airways expressed CFTR on ciliated and nonciliated cells, induced anion transport, and improved the airway surface liquid pH and bacterial killing. Most gene therapy studies to date focus solely on Cl^– transport as the primary metric of phenotypic correction. Here, we describe a gene therapy experiment where we not only correct defective anion transport, but also restore bacterial killing in CFTR-null pig airways.

Authors

Benjamin Steines, David D. Dickey, Jamie Bergen, Katherine J.D.A. Excoffon, John R. Weinstein, Xiaopeng Li, Ziying Yan, Mahmoud H. Abou Alaiwa, Viral S. Shah, Drake C. Bouzek, Linda S. Powers, Nicholas D. Gansemer, Lynda S. Ostedgaard, John F. Engelhardt, David A. Stoltz, Michael J. Welsh, Patrick L. Sinn, David V. Schaffer, Joseph Zabner

Lentiviral-mediated phenotypic correction of cystic fibrosis pigs

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Abstract

Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutations in CF transmembrane conductance regulator (CFTR), resulting in defective anion transport. Regardless of the disease-causing mutation, gene therapy is a strategy to restore anion transport to airway epithelia. Indeed, viral vector–delivered CFTR can complement the anion channel defect. In this proof-of-principle study, functional in vivo CFTR channel activity was restored in the airways of CF pigs using a feline immunodeficiency virus–based (FIV-based) lentiviral vector pseudotyped with the GP64 envelope. Three newborn CF pigs received aerosolized FIV-CFTR to the nose and lung. Two weeks after viral vector delivery, epithelial tissues were analyzed for functional correction. In freshly excised tracheal and bronchus tissues and cultured ethmoid sinus cells, we observed a significant increase in transepithelial cAMP-stimulated current, evidence of functional CFTR. In addition, we observed increases in tracheal airway surface liquid pH and bacterial killing in CFTR vector–treated animals. Together, these data provide the first evidence to our knowledge that lentiviral delivery of CFTR can partially correct the anion channel defect in a large-animal CF model and validate a translational strategy to treat or prevent CF lung disease.

Authors

Ashley L. Cooney, Mahmoud H. Abou Alaiwa, Viral S. Shah, Drake C. Bouzek, Mallory R. Stroik, Linda S. Powers, Nick D. Gansemer, David K. Meyerholz, Michael J. Welsh, David A. Stoltz, Patrick L. Sinn, Paul B. McCray Jr.

A modifier screen identifies DNAJB6 as a cardiomyopathy susceptibility gene

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Abstract

Mutagenesis screening is a powerful forward genetic approach that has been successfully applied in lower-model organisms to discover genetic factors for biological processes. This phenotype-based approach has yet to be established in vertebrates for probing major human diseases, largely because of the complexity of colony management. Herein, we report a rapid strategy for identifying genetic modifiers of cardiomyopathy (CM). Based on the application of doxorubicin stress to zebrafish insertional cardiac (ZIC) mutants, we identified 4 candidate CM-modifying genes, of which 3 have been linked previously to CM. The long isoform of DnaJ (Hsp40) homolog, subfamily B, member 6b (dnajb6b(L)) was identified as a CM susceptibility gene, supported by identification of rare variants in its human ortholog DNAJB6 from CM patients. Mechanistic studies indicated that the deleterious, loss-of-function modifying effects of dnajb6b(L) can be ameliorated by inhibition of ER stress. In contrast, overexpression of dnajb6(L) exerts cardioprotective effects on both fish and mouse CM models. Together, our findings establish a mutagenesis screening strategy that is scalable for systematic identification of genetic modifiers of CM, feasible to suggest therapeutic targets, and expandable to other major human diseases.

Authors

Yonghe Ding, Pamela A. Long, J. Martijn Bos, Yu-Huan Shih, Xiao Ma, Rhianna S. Sundsbak, Jianhua Chen, Yiwen Jiang, Liqun Zhao, Xinyang Hu, Jianan Wang, Yongyong Shi, Michael J. Ackerman, Xueying Lin, Stephen C. Ekker, Margaret M. Redfield, Timothy M. Olson, Xiaolei Xu

Endothelial and circulating C19MC microRNAs are biomarkers of infantile hemangioma

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Abstract

Infantile hemangioma (IH) is the most common vascular tumor of infancy, and it uniquely regresses in response to oral propranolol. MicroRNAs (miRNAs) have emerged as key regulators of vascular development and are dysregulated in many disease processes, but the role of miRNAs in IH growth has not been investigated. We report expression of C19MC, a primate-specific megacluster of miRNAs expressed in placenta with rare expression in postnatal tissues, in glucose transporter 1–expressing (GLUT-1–expressing) IH endothelial cells and in the plasma of children with IH. Tissue or circulating C19MC miRNAs were not detectable in patients having 9 other types of vascular anomalies or unaffected children, identifying C19MC miRNAs as the first circulating biomarkers of IH. Levels of circulating C19MC miRNAs correlated with IH tumor size and propranolol treatment response, and IH tissue from children treated with propranolol or from children with partially involuted tumors contained lower levels of C19MC miRNAs than untreated, proliferative tumors, implicating C19MC miRNAs as potential drivers of IH pathogenesis. Detection of C19MC miRNAs in the circulation of infants with IH may provide a specific and noninvasive means of IH diagnosis and identification of candidates for propranolol therapy as well as a means to monitor treatment response.

Authors

Graham M. Strub, Andrew L. Kirsh, Mark E. Whipple, Winston P. Kuo, Rachel B. Keller, Raj P. Kapur, Mark W. Majesky, Jonathan A. Perkins

Enhanced protective antibody to a mutant meningococcal factor H-binding protein with low-factor H binding

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Abstract

Meningococcal factor H-binding protein (FHbp) is an antigen in 2 serogroup B meningococcal vaccines. FHbp specifically binds human and some nonhuman primate complement FH. To investigate the effect of binding of FH to FHbp on protective antibody responses, we immunized infant rhesus macaques with either a control recombinant FHbp antigen that bound macaque FH or a mutant antigen with 2 amino acid substitutions and >250-fold lower affinity for FH. The mutant antigen elicited 3-fold higher serum IgG anti-FHbp titers and up to 15-fold higher serum bactericidal titers than the control FHbp vaccine. When comparing sera with similar IgG anti-FHbp titers, the antibodies elicited by the mutant antigen gave greater deposition of complement component C4b on live meningococci (classical complement pathway) and inhibited binding of FH, while the anti-FHbp antibodies elicited by the control vaccine enhanced FH binding. Thus, the mutant FHbp vaccine elicited an anti-FHbp antibody repertoire directed at FHbp epitopes within the FH binding site, which resulted in greater protective activity than the antibodies elicited by the control vaccine, which targeted FHbp epitopes outside of the FH combining site. Binding of a host protein to a vaccine antigen impairs protective antibody responses, which can be overcome with low-binding mutant antigens.

Authors

Dan M. Granoff, Serena Giuntini, Flor A. Gowans, Eduardo Lujan, Kelsey Sharkey, Peter T. Beernink

Vascular mineralocorticoid receptor regulates microRNA-155 to promote vasoconstriction and rising blood pressure with aging

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Abstract

Hypertension is nearly universal yet poorly controlled in the elderly despite proven benefits of intensive treatment. Mice lacking mineralocorticoid receptors in smooth muscle cells (SMC-MR-KO) are protected from rising blood pressure (BP) with aging, despite normal renal function. Vasoconstriction is attenuated in aged SMC-MR-KO mice, thus they were used to explore vascular mechanisms that may contribute to hypertension with aging. MicroRNA (miR) profiling identified miR-155 as the most down-regulated miR with vascular aging in MR-intact but not SMC-MR-KO mice. The aging-associated decrease in miR-155 in mesenteric resistance vessels was associated with increased mRNA abundance of MR and of predicted miR-155 targets Cav1.2 (L-type calcium channel (LTCC) subunit) and angiotensin type-1 receptor (AgtR1). SMC-MR-KO mice lacked these aging-associated vascular gene expression changes. In HEK293 cells, MR repressed miR-155 promoter activity. In cultured SMCs, miR-155 decreased Cav1.2 and AgtR1 mRNA. Compared to MR-intact littermates, aged SMC-MR-KO mice had decreased systolic BP, myogenic tone, SMC LTCC current, mesenteric vessel calcium influx, LTCC-induced vasoconstriction and angiotensin II-induced vasoconstriction and oxidative stress. Restoration of miR-155 specifically in SMCs of aged MR-intact mice decreased Cav1.2 and AgtR1 mRNA and attenuated LTCC-mediated and angiotensin II-induced vasoconstriction and oxidative stress. Finally, in a trial of MR blockade in elderly humans, changes in serum miR-155 predicted the BP treatment response. Thus, SMC-MR regulation of miR-155, Cav1.2 and AgtR1 impacts vasoconstriction with aging. This novel mechanism identifies potential new treatment strategies and biomarkers to improve and individualize antihypertensive therapy in the elderly.

Authors

Jennifer J. DuPont, Amy McCurley, Ana P. Davel, Joseph McCarthy, Shawn B. Bender, Kwangseok Hong, Yan Yang, Jeung-Ki Yoo, Mark Aronovitz, Wendy E. Baur, Demetra D. Christou, Michael A. Hill, Iris Z. Jaffe

Multiparametric profiling of non–small-cell lung cancers reveals distinct immunophenotypes

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Abstract

BACKGROUND. Immune checkpoint blockade improves survival in a subset of patients with non–small-cell lung cancer (NSCLC), but robust biomarkers that predict response to PD-1 pathway inhibitors are lacking. Furthermore, our understanding of the diversity of the NSCLC tumor immune microenvironment remains limited.

METHODS. We performed comprehensive flow cytometric immunoprofiling on both tumor and immune cells from 51 NSCLCs and integrated this analysis with clinical and histopathologic characteristics, next-generation sequencing, mRNA expression, and PD-L1 immunohistochemistry (IHC).

RESULTS. Cytometric profiling identified an immunologically “hot” cluster with abundant CD8⁺ T cells expressing high levels of PD-1 and TIM-3 and an immunologically “cold” cluster with lower relative abundance of CD8⁺ T cells and expression of inhibitory markers. The “hot” cluster was highly enriched for expression of genes associated with T cell trafficking and cytotoxic function and high PD-L1 expression by IHC. There was no correlation between immunophenotype and KRAS or EGFR mutation, or patient smoking history, but we did observe an enrichment of squamous subtype and tumors with higher mutation burden in the “hot” cluster. Additionally, approximately 20% of cases had high B cell infiltrates with a subset producing IL-10.

CONCLUSIONS. Our results support the use of immune-based metrics to study response and resistance to immunotherapy in lung cancer.

FUNDING. The Robert A. and Renée E. Belfer Family Foundation, Expect Miracles Foundation, Starr Cancer Consortium, Stand Up to Cancer Foundation, Conquer Cancer Foundation, International Association for the Study of Lung Cancer, National Cancer Institute (R01 CA205150), and the Damon Runyon Cancer Research Foundation.

Authors

Patrick H. Lizotte, Elena V. Ivanova, Mark M. Awad, Robert E. Jones, Lauren Keogh, Hongye Liu, Ruben Dries, Christina Almonte, Grit S. Herter-Sprie, Abigail Santos, Nora B. Feeney, Cloud P. Paweletz, Meghana M. Kulkarni, Adam J. Bass, Anil K. Rustgi, Guo-Cheng Yuan, Donald W. Kufe, Pasi A. Jänne, Peter S. Hammerman, Lynette M. Sholl, F. Stephen Hodi, William G. Richards, Raphael Bueno, Jessie M. English, Mark A. Bittinger, Kwok-Kin Wong

Analysis of cystic fibrosis–associated P67L CFTR illustrates barriers to personalized therapeutics for orphan diseases

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Abstract

Emerging knowledge indicates the difficulty in categorizing unusual cystic fibrosis (CF) mutations, with regard to both pathogenic mechanism and theratype. As case in point, we present data concerning P67L mutation of the cystic fibrosis transmembrane conductance regulator (CFTR), a defect carried by a small number of individuals with CF and sometimes attributed to a channel conductance abnormality. Findings from our laboratory and others establish that P67L causes protein misfolding, disrupts maturation, confers gating defects, is thermally stable, and exhibits near normal conductance. These results provide one framework by which rare CF alleles such as P67L can be more comprehensively profiled vis-à-vis molecular pathogenesis. We also demonstrate that emerging CF treatments — ivacaftor and lumacaftor — can mediate pronounced pharmacologic activation of P67L CFTR. Infrequent CF alleles are often improperly characterized, in part, due to the small numbers of patients involved. Moreover, access to new personalized treatments among patients with ultra-orphan genotypes has been limited by difficulty arranging phase III clinical trials, and off-label prescribing has been impaired by high drug cost and difficulty arranging third party reimbursement. Rare CFTR mutations such as P67L are emblematic of the challenges to “precision” medicine, including use of the best available mechanistic knowledge to treat patients with unusual forms of disease.

Authors

Carleen M. Sabusap, Wei Wang, Carmel M. McNicholas, W. Joon Chung, Lianwu Fu, Hui Wen, Marina Mazur, Kevin L. Kirk, James F. Collawn, Jeong S. Hong, Eric J. Sorscher