An important discovery in 1949 by Pauling (Pauling L et al. Science) and biophysical validation 8 years later by Ingram (Ingram VM. Nature 1957) relating to the β-globin chain position 6 glutamic acid>valine substitution initiating sickling and leading to haemoglobin tetramerization (α2βs2) laid the genetic foundation for understanding sickle cell disease or SCD. High prevalence of the disease is primarily attributed to carriers from high malaria regions, and the impact of SCD increases with high mortality rates. Earlier treatments like the oral administration of hydroxylusea (HU), a chemotherapeutic agent used for chronic myeloid leukaemia and polycythemia vera, has to some extent increased the HbF levels. In 1998, the US Food and Drug Administration (FDA) gave its approval after HU showed a modest increase in levels of HbF in primates and many patients with SCD (Platt OS, et al. J. Clin. Investig 1984). However, the mechanism was not cleared to be used as a sustainable therapy system. This was also due to the fact that the drug does not correct the underlying genetic abnormality, but only increases the red blood cell size for temporary relief. To reverse the underlying pathophysiology, it is important to block the polymerization or increase non-sickling haemoglobin. However, the majority of therapies focus on secondary or tertiary clinical features like inflammation, pain and leukocyte adhesiveness.
SCD affects more than 250,000 new patients every year (Modell B, Darlison M. Bull World Health Organ. 2008) with an average age of 36-40 years (Powars DR, et al. Medicine (Baltimore) 2005 & Delea TE et al. Am J Hematol 2008). Presently the primary therapy available for SCD is allogeneic hematopoietic stem cell (HSC) transplant. However, it is difficult to find fully matching donors. Those who receive mismatched transplants could suffer from several complications associated with graft rejection or graft-versus-host problems.
Gene therapy has the potential for developing better long-term reprieve for patients. For gene therapy to be successful in patients, two major challenges need to be addressed: (1) Gene correction for long term HSC repopulation, and (2) endogenous regulation and stable expression of the new or repaired gene. Initial ϒ-retroviral vector-based gene transfer experiments in mice to achieve high-level erythroid-specific expression of the β-globin gene were not successful to the extent expected (Novak U, et al. Proc Natl Acad Sci USA. 1990 & Karlsson S, et al. Proc Natl Acad Sci USA. 1988 & Dzierzak EA, et al. Nature. 1988). However, the discovery and characterization of the β-globin locus control region (LCR) (Grosveld F, et al. Cell. 1987) helped in identifying the hypersensitive regulatory regions. However, we still need to identify better erythroid-specific enhancer elements. The other challenges include expression variegation, silencing, changing the expression of neighboring genes and enhancer-blocking activity.
With the advent of new genome editing technologies, better therapeutic options have been evolving. Targeted nucleases like zinc-finger nucleases (ZNFs), transcription-activators like effector nucleases (TALENs), meganucleases and CRISPR (clustered, regulatory interspaced palindromic repeats) associated nuclease Cas9, which can introduce site specific changes in the genome, have become valuable. Unlike gene addition programmes, gene editing has now become a better way to work towards therapeutic strategies. In principle, this approach requires the nuclease for the double strand break and repair template for correction based on homologous DNA repair mechanism (Voit RA, Nucleic Acids Res. 2014). Several new methods, such as oligonucleotide-based gene therapy strategies and triplex forming peptide nucleic acids have been used, but with a very low yield of correction of SCD in HSCs. A similar approach, using non-homologous end joining (NHEJ) repair in HSCs, was observed to correct SCID-X1 mutation in human HSCs (Genovese P, et al. Nature. 2014), suggesting that the HDR (homology-driven repair) pathway is restricted to S and G2 phase when sister chromatids are available for repair.
In comparison to HDR, NHEJ seems to be a more robust repair mechanism as shown in the clinical trials for gene disruption in T cells using ZNF targeting CCR5 in HIV-infected subjects, demonstrating that it is safe and efficacious (Tebas P, et al. N Engl J Med. 2014). A better understanding of the mechanisms of globin gene regulation can give alternative strategies to repair. These approaches need not be gene addition or correction but can target a modifier such as elevated HbF levels in SCD. Genome-wide association studies have identified 3 important loci — BCL11A, the intergenic region between HBS1L and MYB and β-globin cluster regulating HbF level (Menzel S, et al. Nat Genet. 2007 & Uda M, et al. Proc Natl Acad Sci USA. 2008). In transgenic mice, the BCL11A region showed an important role in the regulation of HbF levels (Sankaran VG, et al. Science. 2008 & Xu J, et al. Science. 2011). The data suggest that BCL11A loss was sufficient in mice to reverse haematologic and pathological manifestations of the disease and suggests that BCL11A is a potentially good therapeutic target. Similar to BCL11A, BTB-family proteins LRF/ZBTB7A seem to play a role in the repression of HbF expression (Masuda T, et al. Science 2016). It was shown that the combined effect of Lrf and BCL11A was better in silencing HbF and they both interact with nucleosome remodeling and deacetylase (NuRD).
Gene addition of β-globin, on the other hand, aims to obtain HSCs which are genetically modified ex vivo and returned to the patient, potentially resulting in lifelong hematopoietic autograft to produce modified red cells. After nearly two decades of preclinical studies, clinical trials (Ribeil J-A, N. Engl. J. Med. 2017) using a vector carrying an antisickling β-globin variant T87Q was studied. Of the 8 patients with severe genotype β0β0, six remained transfusion dependent and two were anaemic with haemoglobin levels of 9-10g/dL. Out of the nine milder patients with βEβ0 genotype, two were able to stop transfusions and maintained 9-10mg/dL haemoglobin. The critical factor for better clinical outcomes seems to be the degree of gene transfer or vector copy number in the infused cells. There are 12 ongoing clinical trials for SCD, which are recruiting participants, of which four (NCT02186418, NCT03282656, NCT02247843, NCT02140554) are evaluating the efficacy and safety of gene therapy where a mutated β-globin gene is replaced with a normal, functional wild type gene (United States and Jamaica). Three trials (NCT02193191, NCT02989701, NCT03226691) are evaluating the value of Mozobil (plerixafor) in SCD patients for increasing the production of stem cells for gene therapy. Other studies are focused on enabling a better understanding of the therapy modules. Gene repair or editing based therapeutic mechanisms are still under study and early reports are promising, but seem to be very low in efficacy.
With careful attention, improvements and experimentation, gene correction could play a major role in therapy. Recent FDA approval of CRISPR based CTX001 gene therapy for stem cells derived from the same patient has started. Such reports suggest that new therapeutic regimes might be much closer than thought.
The author is medical scientist and former director of SGRF, Bangalore