A dual-acting DNASE1/DNASE1L3 biologic prevents autoimmunity and death in genetic and induced lupus models

A defining feature of systemic lupus erythematosus (SLE) is loss of tolerance to self-DNA, and deficiency of DNASE1L3, the main enzyme responsible for chromatin degradation in blood, is also associated with SLE. This association can be found in an ultrarare population of pediatric patients with DNASE1L3 deficiency who develop SLE, adult patients with loss-of-function variants of DNASE1L3 who are at a higher risk for SLE, and patients with sporadic SLE who have neutralizing autoantibodies against DNASE1L3. To mitigate the pathogenic effects of inherited and acquired DNASE1L3 deficiencies, we engineered a long-acting enzyme biologic with dual DNASE1/DNASE1L3 activity that is resistant to DNASE1 and DNASE1L3 inhibitors. Notably, we found that the biologic prevented the development of lupus in Dnase1–/–Dnase1L3–/– double-knockout mice and rescued animals from death in pristane-induced lupus. Finally, we confirmed that the human isoform of the enzyme biologic was not recognized by autoantibodies in SLE and efficiently degraded genomic and mitochondrial cell–free DNA, as well as microparticle DNA, in SLE plasma. Our findings suggest that autoimmune diseases characterized by aberrant DNA accumulation, such as SLE, can be effectively treated with a replacement DNASE tailored to bypass pathogenic mechanisms, both genetic and acquired, that restrict DNASE1L3 activity.

Analytical HPLC to assess biologic purity: Protein sample (15µg in PBS) was dried at 25°C in SpeedVac for 20 minutes and reconstituted in 30 µL with 95% water 5% acetonitrile and 0.1% trifluoracetic acid.LC-PDA analysis was performed on a Waters H-Class UPLC system (Waters Technologies) utilizing a quaternary solvent system (Buffer A: 99.9% water, 0.1% trifluoracetic acid; Buffer B: 28.6% water, 71.4% acetonitrile, 0.075% trifluoracetic acid).Protein was profiled using an ACQUITY UPLC Protein BEH C4 Column 1.7 µm, 2.1 mm x 150 mm (40°C) and eluted at 0.4 mL/min with the following gradient: 28% buffer B at initial conditions, 100% B at 40 min; maintain 5 min, return to initial conditions at 50 minutes and maintained 20 minutes.The protein was detected in PDA detector with 220 nm channel.

Size exclusion -light scattering chromatography:
The light scattering data were collected using a Superdex S-200, 10/30, HR Size Exclusion Chromatography column (GE Healthcare), connected to an HPLC system, Alliance 2965, (Waters Corporation) equipped with an autosampler.The elution from SEC was monitored by a photodiode array (PDA) UV/VIS detector (996 PDA, Waters Corporation), differential refractometer (OPTI-Lab, or OPTI-rEx Wyatt Corporation,), and static, multiangle laser light scattering detector (DAWN-EOS, Wyatt Corporation).The SEC-UV/LS/RI system was equilibrated in PBS at a flow rate of 0.5 ml/min.
The weight average molecular mass, Mw, for the protein sample was tested at two concentrations of 0.59 mg/ml and 0.08 g/ml (1/20 th dilution).Two software packages were used for data collection and analysis: the Millennium software (Waters Corporation) controlled the HPLC operation and data collection from the multi-wavelength UV/VIS detector, while the ASTRA software (Wyatt Corporation) collected data from the refractive index detector, the light scattering detectors, and recorded the UV trace at 280 nm, 295 nm, or 310 nm sent from the PDA detector; 295 and 310 nm were used for monitoring the elution of protein-DNA complexes, or for protein alone for when A280 >1.
Generation of Dnase1/Dnase1L3-double-deficient (DKO) mice.The Dnase1L3 cKO mouse model was generated via CRISPR-Cas9 methods (1)(2)(3).Potential Cas9 target guide (protospacer) sequences in introns 3 and 4 were screened using the online tool CRISPOR (http://crispor.tefor.net)(4), and candidates were selected.Templates for sgRNA synthesis were generated by PCR, and sgRNAs were transcribed in vitro and purified (Megashortscript, MegaClear; ThermoFisher).sgRNA/Cas9 RNPs were complexed and tested for activity by zygote electroporation, incubation of embryos to blastocyst stage, and genotype scoring of indel creation at the target sites.The sgRNAs that demonstrated the highest activity were selected for creating the floxed allele.Guide RNA (gRNA) sequences are as follows: 5' guide: TCCAGAGCAGGCTCAAGTGG and 3' guide ACCTGACACAAATACCTTGG. Accordingly, a 550 base long single-stranded DNA (lssDNA) recombination template incorporating the 5' and 3' loxP sites was synthesized (Integrated DNA Technologies).The injection mix of sgRNA/Cas9 RNP + lssDNA was microinjected into the pronuclei of C57Bl/6J zygotes (3).Embryos were transferred to the oviducts of pseudo pregnant CD-1 foster females using standard techniques (5).Genotype screening of tissue biopsies from founder pups was performed by PCR amplification and Sanger sequencing to verify the floxed allele.Germline transmission of the correctly targeted allele (i.e., both loxP sites in cis) was confirmed by breeding and sequence analysis.Subsequently, positive mice were mated with a Beta Actin CRE mouse to generate constitutive Dnase1L3-deficient mice.Neutrophil Extracellular Trap (NET) digestion assay.Neutrophils were purified from mouse bone marrow as previously described (6).In brief, bone marrow was flushed from the mouse femur with a 26g needle filled using RPMI supplemented with 10% FBS and 2mM EDTA and passed through a 70uM cell strainer and cells collected by centrifugation at 450 x g for 7min at 4°C. Cell pellet was resuspended in 20 ml of 0.2% NaCl for approximately 20 seconds followed by addition of 20 ml of 1.6% NaCl, followed by centrifugation at 450xg for 7min at 4°C.After .1(KOMP)Vlcg/TcpMuncd, RRID:MMRRC_047412-UCD was obtained from the Mutant Mouse Resource and Research Center (MMRRC) at the University of California, Davis.Dnase1-deficient mice were mated with Dnase1L3-deficient mice to generate the Dnase1/Dnase1L3-double deficient mice, hereafter referred to as DKO mice.
washing cells again in RPMI, the cells were resuspended in 1mL PBS and layered on top of a Histopaque 1119/1077 density gradient, spun at 872 x g for 30 min without break and neutrophils were collected from the interface, washed with RPMI without supplement, plated out into 96 wells cell culture plate at 2.5 x 10(5) cells per well and incubated at 37°C in 5% CO2.NETs were induced by the addition of 50nM PMA with or without a serial dilution from 25-0.325 nM of either Roche DNASE1, LBme, or 1833.After 4 hours, 5uM cell impermeable DNA binding Sytox Green (ThermoFisher Scientific) was added and NETs were visualized on a BZ-X Keyence fluorescence microscope with 488nm laser light and emission collection at 449-552 nm.The level of Sytox Green fluorescence was quantitated on a Synergy Mx microplate reader (BioTek).