Alpha-1-antitrypsin (AT) deficiency and the serpinopathies: pathobiology and new therapeutic strategies

Antitrypsin is found at high concentrations in the bloodstream, where its main role is to protect the lungs against tissue damage from inflammation. Liver cells (hepatocytes) normally release individual molecules of antitrypsin into the circulation. Antitrypsin deficiency results when an individual inherits two genes with small changes in the antitrypsin protein. The Z variant, found in about 4% of people of North European decent, is the most common cause of severe antitrypsin deficiency. The Z variant causes antitrypsin to form long chains of linked molecules (called "polymers") that are trapped inside liver cells. The build-up of polymers damages the cell and increases the chance of developing liver cirrhosis and liver cancer. The reduced amount of properly formed protein in the circulation means the lungs are not as well protected against inflammation and so individuals develop emphysema. We have shown that a similar process occurs in mutants of other members of the protein family to which antitrypsin belongs. These include polymerisation of mutants of neuroserpin in the brain to cause dementia. We have grouped all the conditions together as a single class of disease that we have called 'the serpinopathies'. The application builds on 25 years of work by our group and has 5 interlinking projects within a programme of work. We propose to:

(i) define the structure of the pathological polymer using biophysical analysis and cryo-electron microscopy. We will define the structure of the pathological polymer isolated from human tissues and use this information to develop strategies to block the abnormal protein-protein linkage that underlies antitrypsin deficiency and the serpinopathies.

(ii) establish the cellular response to intracellular serpin polymers. We will use the imaging of cells that express polymers to define the effect of polymer formation on cell function. We will also establish whether cell dysfunction can be reversed by small molecules that block polymer formation and so determine whether it is the polymers themselves that cause cell dysfunction and disease.

(iii) define disease mechanism in a multicellular context and identify new therapeutic targets using a worm (C. elegans) model of antitrypsin deficiency. We have developed a novel worm (C. elegans) model in which Z antitrypsin is retained as polymers in association with impaired motility, thin body structure and delayed development. We will assess the effect of small molecules and polymer blocking antibodies on intracellular polymerisation and the motility of the worm, dissect the mechanism by which intercellular antitrypsin polymers signal toxicity within the cell and undertake a chemical screen for novel agents that can reduce the toxicity of polymers in the worm. These will be used as the basis to design new therapies for use in man.

(iv) define the dynamics of antitrypsin polymers in vivo and their utility as a biomarker of disease. We will study individuals with antitrypsin deficiency undergoing liver and lung transplantation to evaluate the changes in antitrypsin polymers within the lung and circulation. We will also evaluate circulating polymers as the first biomarker that is specific for predicting and diagnosising antitrypsin deficiency related liver disease.

(v) develop a diagnostic technology for imaging Z antitrypsin polymers in vivo. We will use cell penetrating monoclonal antibodies or small molecules that are specific for polymers to image intracellular polymers in mouse models of disease. Our longer term aim is to develop this for use in man so we can assess whether there is a correlation between polymer burden and liver disease and if this new imaging test will be useful to accelerate drug development in man.

Taken together this work will increase our understanding of mechanism of antitrypsin deficiency and the serpinopathies and allow the development of new approaches to treatment.

We described the polymerisation of mutants that underlie antitrypsin deficiency and developed new paradigms for the liver and lung disease associated with this condition. We have also described the polymerisation of mutants of antichymotrypsin that have been associated with COPD and mutants of neuroserpin that cause a novel dementia that we called familial encephalopathy with neuroserpin inclusion bodies (FENIB). We grouped these diseases with others that result from polymerisation of mutants of the serpins as the serpinopathies. We have used biochemistry, biophysical analysis, crystallography, NMR, cell and induced pluripotent stem cells, monoclonal antibodies and Drosophila models of disease to dissect the structural basis of polymer formation and the cellular consequences. We are in late lead optimisation stage of developing small molecules that block polymerisation with GlaxoSmithKline. However it is critical that we understand the pathobiology of antitrypsin deficiency and serpinopathies in more detail in order to evaluate a broad range of therapeutic strategies with the aim of curing the disease. The aims of this programme are to: (i) define the structure of the pathological polymer using biophysical analysis and cryo-electron microscopy, (ii) establish the cellular response to intracellular serpin polymers, (iii) define disease mechanism in a multicellular context and identify new therapeutic targets using a C. elegans model of antitrypsin deficiency, (iv) define the dynamics of antitrypsin polymers in vivo and their utility as a biomarker of disease, (v) develop a diagnostic technology for imaging Z antitrypsin polymers in vivo. The work is enabled by the facilities at UCL and the shared lab in which the applicants work. Indeed the preliminary data have been generated following the move of Lomas and Sattelle to UCL. The findings from this programme are likely to be applicable to a broad range of other protein conformational diseases.

There are many potential impacts of our work:

(i) Our work has led to one of only 8 'Discovery Partnership with Academia' (DPAc) agreements with GlaxoSmithKline to develop small molecules to treat antitrypsin deficiency. This has progressed to the late lead optimisation stage of drug discovery. There is still a long way to go and the proposed programme of work will provide enabling information for the small molecule campaign. However if it is successful then we will develop the first therapy for antitrypsin deficiency that aims to block intracellular polymerisation and increase the secretion of active protein. Moreover the work will provide a paradigm to target other intra-endoplasmic reticulum protein folding diseases. Our work has led to a BBSRC/GSK CASE Studentship to Sarah Faull and an EPSRC/GSK CASE Studentship to Alistair Jagger.

(ii) We have used antitrypsin deficiency as a test system to develop human induced pluripotent stem cell (hIPSCs) models of inherited metabolic liver disorders (that also includes glycogen storage disease Type 1a, familial hypercholesterolaemia, hereditary tyrosinaemia and Crigler Najjar syndrome). This has been used to spin out a company from the University of Cambridge (http://www.definigen.com) by Ludovic Vallier and one of our former PhD students (Tamir Rashid). One of my post-doctoral fellows (Adriana Ordóñez) worked for the company to generate cell lines for commercialisation.

(iii) The novel reagents generated by our programme (eg C. elegans and cell models) will be made available to the academic community and monoclonal antibodies will be commercialised as we have done for the 2C1 monoclonal antibody with Hycult Biotech (http://www.hycultbiotech.com/hm2289).

(iv) Our 2C1 monoclonal antibody detects circulating polymers of antitrypsin. We have shown that Individuals with a wide range of antitrypsin deficiency genotypes have circulating polymers (Eur. Resp. J., 2014;43:1501-1504). We are exploring with Grifols whether the 2C1 monoclonal antibody can be used as a 'point of care' screening test to identify individuals with antitrypsin deficiency. The advantage is that this approach will detect many mutants that cause antitrypsin deficiency whilst the existing test detects only the Z allele.

(v) The cell penetrating monoclonal antibodies exploit technology developed at UCL (http://www.thiologics.com) and will be developed to image polymers within the liver of individuals with antitrypsin deficiency. It is possible that they may also be used to deliver polymer blocking antibodies to the livers of individuals with antitrypsin deficiency. If effective then they will be commercialised through UCL Business (UCLB). It is possible that this approach could be developed to target other intracellular disease processes such as the tauopathies.

(vi) Our work has an immediate impact on patient care as we characterise the scientific and clinical significance of antitrypsin deficiency mutations identified as part of clinical service that is now at the Royal Free Hospital (eg. Hepatology 2010; 52: 1078-1088; Am. J. Resp. Cell Mol Biol. 2015 In press). We have also described the prevalence and risk factors for liver involvement in individuals with PiZZ antitrypsin related lung disease (Am. J. Resp. Crit. Care Med. 2013; 187: 502-508).

(vii) If successful circulating antitrypsin polymers would be the first biomarker that is specific for antitrypsin deficiency related liver disease.

(viiI) If one of our therapeutic approaches is successful then this would provide a rational to screen newborn infants for antitrypsin deficiency to prevent them developing established liver disease. This is not currently undertaken in the UK as it is argued that there is no therapeutic benefit from early diagnosis.