Fusion proteins targeted to the central nervous system

Abstract

The present invention relates to fusion proteins targeted to the central nervous system (CNS) and their use for the treatment of lysosomal storage diseases (LSDs).

Description

[Technical field]

[0001] The present invention relates to fusion proteins targeted to the central nervous system (CNS) and their use for the treatment of lysosomal storage diseases (LSDs). [Background technology]

[0002] Lysosomes host over 60 soluble lysosomal hydrolases and accessory proteins, as well as over 120 lysosomal membrane proteins and transient protein residues. 1 Dysfunction in some of these proteins collectively results in lysosomal storage disorders (LSDs) that have a relatively high incidence in the general population, with more than 1:5000 births being affected by LSDs. 2 Among these lysosomal enzymes, β-glucocerebrosidase (GCase), encoded by GBA and responsible for the hydrolytic release of glucose from the glycolipids glucosylsphingosine (GlcSph) and glucosylceramide (GlcCer), is noteworthy. Deficiency in GCase enzymatic activity leads to the accumulation of these glycolipids causing lysosomal storage diseases with various disease severity. 3 Neuropathic Gaucher disease (GD), type 2, is caused by severe or null mutations in GBA1 and is ultimately fatal. Currently, there are no drugs that can modify the disease course. 4~7 Although often debilitating, chronic neuropathic or type 3 GD has varying degrees of neurological symptoms, but patients may survive infancy and develop symptoms later in life. 8 Both homozygous and heterozygous carriers of the mutant GBA1 allele are at high risk for sporadic and complex neurodegenerative diseases, including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Again, no disease-modifying therapies are available to slow the disease course. GBA1-associated PD (GBA-PD) is neuropathologically indistinguishable from sporadic PD, but is often associated with earlier disease onset, more prominent non-motor symptoms, and faster disease progression. 9、10Treatment paradigms targeting impaired GCase to restore its intracellular lysosomal function are expected to prove beneficial in disorders ranging from neuropathic GD to neurodegenerative diseases such as PD and DLB.

[0003] GD- and PD-associated pathological variants in GBA1, such as L444P (p.L483P) and N370S (p.N409P), result in the production of misfolded mutant enzymes with significantly reduced activity, ranging from 10-20% of normal. 11 Accumulation of glycosphingolipids due to reduced GCase activity may be a key pathological event in GD and a precipitating event for neurodegeneration associated with PD. 12~14 Therefore, taking into account the common genetics, increasing GCase lysosomal activity in the brain may very well be a viable therapeutic approach to restore lysosomal homeostasis in both neuronopathic GD and GBA1-related neurodegenerative diseases.

[0004] Currently available treatments for GD include substrate reduction therapy using small molecules or enzyme replacement therapy (ERT). However, neither can target GCase deficiency in the CNS. Therefore, there is a need for molecules and therapies that target the CNS aspects of LSD. Summary of the Invention

[0005] In a first aspect, the present invention provides a fusion protein comprising a lysosomal protein, an Fc region of an antibody, and an antibody fragment that targets the transferrin receptor, wherein the antibody fragment has a monovalent binding mode.

[0006] In one embodiment of the invention, the lysosomal protein is a β-glucocerebrosidase (Gcase) protein, preferably a human Gcase protein or a variant thereof.

[0007] In one embodiment of the invention, the Fc region of an antibody is an Fc region of an IgG antibody, preferably an IgG1 antibody.

[0008] In one embodiment of the invention, the Fc region lacks Fc receptor gamma binding.

[0009] In certain embodiments of the invention, antibody fragments that target the transferrin receptor include Fv, Fab, Fab', Fab'-SH, F(ab') 、 It is selected from the group consisting of diabodies, linear antibodies, single chain antibody molecules such as scFv, scFab, cross-Fab and single domain antibodies (dAb).

[0010] In one embodiment of the invention, one chain of the Fc region is fused at its N-terminus to the C-terminus of a lysosomal protein and a second Fc chain is fused at its C-terminus to an antibody fragment that targets the transferrin receptor.

[0011] In one embodiment of the invention, the two Fc chains form a dimer using knob-into-hole technology.

[0012] In one embodiment of the invention, the fusion protein comprises two protein chains: 1. a first protein chain comprising a lysosomal protein fused at its C-terminus to a first chain of an Fc region comprising knob-into-hole technology; 2. A second protein chain comprising a second chain of an Fc region containing knobs-into-hole technology fused at its C-terminus to an scFab antibody fragment targeting the transferrin receptor.

[0013] In one embodiment of the invention, the human Gcase protein has the amino acid sequence shown in SEQ ID NO:1.

[0014] In one embodiment of the invention, the first protein chain has the amino acid sequence set forth in SEQ ID NO:2 and the second single chain protein has the amino acid sequence set forth in SEQ ID NO:3.

[0015] In a second aspect, the present invention relates to an isolated nucleic acid molecule encoding the fusion protein of the invention.

[0016] In one embodiment of the invention, the nucleic acid is circular RNA.

[0017] In a third aspect, the present invention relates to a host cell comprising the isolated nucleic acid molecule of the present invention.

[0018] In a fourth aspect, the present invention provides a pharmaceutical formulation comprising a fusion protein of the invention.

[0019] In a further aspect, the present invention provides the fusion protein of the invention as a medicament.

[0020] In a further aspect, the present invention relates to the use of the fusion protein of the invention for the treatment of neurodegenerative disorders, in particular LSDs, more particularly CNS aspects of LSDs.

[0021] In a further aspect, the present invention relates to a recombinant AAV vector comprising a nucleic acid molecule encoding the fusion protein of the invention.

[0022] In one embodiment, the invention relates to an AAV viral particle comprising an AAV vector of the invention.

[0023] In one embodiment, the present invention relates to a pharmaceutical composition comprising the AAV viral particle of the present invention.

[0024] In a further embodiment, the present invention provides the use of the AAV viral particle of the invention for the treatment of neurodegenerative disorders, in particular LSDs, more particularly CNS aspects of LSDs.

[0025] definition The term "lysosomal proteins" refers to proteins that are localized to lysosomes, including over 60 soluble lysosomal hydrolases and accessory proteins, as well as over 120 lysosomal membrane proteins and transient protein residues.

[0026] As used herein, "GCase" is an abbreviation used for β-glucocerebrosidase. Human β-glucocerebrosidase has Uniprot ID: P04062. The amino acid sequence of mature human GCase variant R534H is shown in SEQ ID NO: 1. Mature human GCase comprises amino acids 40-536 of human GCase having Uniprot ID: P04062. Furthermore, variants, recombinant β-glucocerebrosidase proteins also include functional fragments or derivatives thereof.

[0027] "Blood-brain barrier" or "BBB" refers to the physiological barrier between the peripheral circulation and the brain and spinal cord (i.e., CNS) formed by tight junctions in the brain capillary endothelial cell membrane, forming a tight barrier that limits the transport of molecules, even very small molecules such as urea (60 daltons), into the brain. The blood-brain barrier in the brain, the blood-spinal cord barrier in the spinal cord, and the blood-retina barrier in the retina are continuous capillary barriers in the CNS, and are collectively referred to herein as the blood-brain barrier or BBB. The BBB also encompasses the blood-CSF barrier (choroid plexus), which is composed of ependymal cells rather than capillary endothelial cells.

[0028] The "transferrin receptor" ("TfR") is a transmembrane glycoprotein (molecular weight approximately 180,000) composed of two disulfide-linked subunits (each with an apparent molecular weight of approximately 90,000) that is involved in iron uptake in vertebrates. In one embodiment, the TfR herein is a human TfR comprising the amino acid sequence set forth in, for example, Schneider et al. Nature 311:675-678 (1984).

[0029] The term "nucleic acid molecule" or "polynucleotide" includes any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Nucleic acid molecules are often described by the sequence of bases, whereby the bases represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is typically represented from 5' to 3'. As used herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules may be linear or circular. In addition, the term nucleic acid molecule includes both sense and antisense strands, and both single-stranded and double-stranded forms.Furthermore, the nucleic acid molecules described herein can include naturally occurring or non-naturally occurring nucleotides.Examples of non-naturally occurring nucleotides include modified nucleotide bases, including derivatized sugar or phosphate backbone bonds or chemically modified residues.Nucleic acid molecules also include DNA and RNA molecules suitable as vectors for direct expression of the antibody of the present invention in vitro and / or in vivo, for example, in a host or patient.Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, the mRNA can be chemically modified to increase the stability of the RNA vector and / or the expression of the encoded molecule, such that the mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler ert al, Nature Medicine 2017, published online June 12, 2017, doi:10.1038 / nm.4356 or European Patent No. 2101823 B1).

[0030] An "isolated" nucleic acid refers to a nucleic acid molecule that is separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that ordinarily contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0031] "Percentage (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing gaps if necessary to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of sequence identity for alignment. Alignment for determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or FASTA program package. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the entire length of the sequences being compared. Alternatively, percent identity values ​​can be generated using sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc. and the source code is on file in the user documentation of the U.S. Copyright Office, Washington DC, 20559, registered under U.S. Copyright Registration No. TXU510087, and described in WO 2001 / 007611.

[0032] Unless otherwise indicated, for purposes herein, percent amino acid sequence identity values ​​are generated using the ggsearch program of the FASTA package version 36.3.8c, or the subsequent BLOSUM50 comparison matrix. The FASTA program package is certified by WR Pearson and DJ Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448; WR Pearson (1996) "Effective protein sequence comparison" Meth. Enzymol. 266:227-258; and Pearson et.al. (1997) Genomics 46:24-36, and is publicly available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, sequences can be compared using the public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi using the ggsearch (global protein:protein) program and default options (BLOSUM50; open:-10; ext:-2; Ktup=2), ensuring a global rather than local alignment. Percent amino acid identity is given in the output alignment header.

[0033] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that is in a form such that the biological activity of the active ingredients contained therein is effective and that does not contain additional ingredients that are unacceptably toxic to a subject to which the pharmaceutical composition may be administered.

[0034] A "pharmaceutically acceptable carrier" refers to an ingredient, other than an active ingredient, in a pharmaceutical composition or formulation that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, additives, stabilizers, or preservatives.

[0035] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and may be performed for prophylaxis or during the course of clinical pathology. The desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, relief of symptoms, attenuation of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing the rate of disease progression, amelioration or palliation of symptoms, and recovery or improved prognosis. In some embodiments, the antibodies of the invention are used to delay disease onset or to slow disease progression.

[0036] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the antibody produced by the host cell may undergo post-translational cleavage of one or more, in particular one or two, amino acids from the C-terminus of the heavy chain. Thus, upon expression of a particular nucleic acid molecule encoding a full-length heavy chain, the antibody produced by the host cell may comprise a full-length heavy chain or may comprise a cleaved variant of the full-length heavy chain. This is the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU numbering system). Thus, the C-terminal lysine (Lys447) of the Fc region, or the C-terminal glycine (Gly446) and lysine (Lys447) may or may not be present. The amino acid sequence of the heavy chain comprising the Fc region is shown herein without the C-terminal glycine-lysine dipeptide, unless otherwise specified. In one aspect, the heavy chain comprising the Fc region as specified herein comprised in the antibody according to the invention comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, EU numbering system). In one aspect, the heavy chain comprising the Fc region as specified herein comprised in the antibody according to the invention comprises an additional C-terminal glycine residue (G446, EU index numbering). Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0037] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005). In certain embodiments, the antibody fragment is an anti-transferrin receptor antibody, such as those disclosed in WO2014 / 033074 and WO2012 / 075037. In certain embodiments, the antibody fragment is a Fab or scFab antibody fragment, preferably a cross-Fab antibody fragment, against the human transferrin receptor. Exemplary cross-Fab fragments are described in WO 2009 / 080251, WO 2009 / 080252 and MABS 2016, VOL. 8, NO. 6, 1010-1020.

[0038] "Monovalent binding mode" refers to specific binding to TfR where the interaction between the antibody fragment and TfR occurs through a single epitope. The monovalent binding mode prevents dimerization / multimerization of TfR due to a single epitope interaction point. The monovalent binding mode prevents the intracellular sorting of TfR from being altered.

[0039] The term "epitope" includes any polypeptide determinant capable of specific binding to an antibody. In certain embodiments, epitopic determinants include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and in certain embodiments may have specific three-dimensional structural characteristics, and / or specific charge characteristics. An epitope is the region of an antigen that is bound by an antibody.

[0040] The term "AAV" is the standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a coinfecting helper virus. Currently, there are 13 characterized serotypes of AAV. General information and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). However, it is well known that the various serotypes are very closely related, both structurally and functionally, even at the genetic level, so it is fully expected that these same principles will be applicable to additional AAV serotypes. (See, e.g., Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J.R. Puttison, ed.; and Rose, Comprehensive Virology 3:1-61 (1974)).

[0041] As used herein, "AAV vector" refers to a vector that contains one or more polynucleotides of interest (or transgenes) flanked by AAV terminal repeats (ITRs). Such AAV vectors can replicate and be packaged into infectious viral particles when present in a host cell transfected with a vector that encodes and expresses the rep and cap gene products.

[0042] "AAV viral particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. When the particle contains a heterologous polynucleotide (i.e., a polynucleotide such as a transgene to be delivered to a mammalian cell), it is typically referred to as an "AAV vector particle" or simply an "AAV vector." [Brief description of the drawings]

[0043] [Figure 1] The purified GCase-BS molecule is fully functional. (A) Schematic of GCase-BS showing the different parts. (B) Evaluation of mouse transferrin receptor (mTfR) binding of mGCase-mBS by FACS analysis of mTfR expressing cells. (C) Evaluation of human transferrin receptor (hTfR) binding of hGCase-hBS by FACS analysis of hTfR expressing cells. The specificity of the TfR binding part was shown for mBS, since no signal was observed in hTfR expressing cells. (D) Enzyme activity (Michaelis-Menten diagram) of the different GCase(-BS) molecules. Resorufin-β-glucopyranoside was used as substrate. (E) Enzyme activity and IgG levels measured for both mGCase-mBS and hGCase-hBS after 15 min. [Diagram 2]The Brain Shuttle module improves cellular uptake and lysosomal efficacy in vitro. (A) Total GCase activity in mouse cortical neurons as a measure of cellular uptake after 2 h treatment with imiglucerase, mGCase or mGCase-mBS. Data was normalized to GBA+ / + cells. (B) Total GCase activity in H4 cells as a measure of cellular uptake after 2 h treatment with Cer, hGCase-hBS-NB or hGCase-hBS. Data was normalized to GBA+ / + cells. (C) Live imaging and quantification of colocalized signals of GCase activity (FQ-7) and lysosomes (SiR lyso) in mouse cortical neurons. Data was normalized to GBA+ / + cells. (D) Live imaging and quantification of colocalized signals of GCase activity (FQ-7) and lysosomes (SiR lyso) in H4 cells. Data was normalized to GBA+ / + cells. (E) Measurement of glucosylsphingosine in mouse cortical neurons as a measure of lysosomal availability after 48 h of treatment with Cer, mGCase or mGCase-mBS. Data were normalized to GBA- / - cells. (F) Measurement of glucosylsphingosine in H4 cells as a measure of lysosomal availability after 48 h of treatment with Cer, hGCase-NB or hGCase-hBS. Data were normalized to GBA- / - cells. Bar graphs represent group mean + SEM. n=3. Activity data were analyzed by two-way ANOVA (Tukey's multiple comparison test). **p<0.01; ***p<0.001; ****p<0.0001. [Diagram 3]GCase-BS lysosomal mode of action in vitro. (A) Immunolabeling of hGCase and colocalization with LAMP1 upon acute treatment with various BS constructs + / - fusions to hGCase. Colocalized hBS spots were quantified and normalized to total LAMP1 spots. n=5. (B) Immunolabeling of hGCase and colocalization with LAMP1 upon acute treatment with various BS constructs + / - fusions to hGCase. Colocalized GCase spots were quantified and normalized to total LAMP1 spots. (C) Total GCase activity in GBA-deficient TfR WT and TfR KO neuroblastoma lines. (D) Glucosylsphingosine measurement in GBA-deficient TfR WT and TfR KO neuroblastoma lines. (E) Total GCase activity in GBA-deficient M6PR-CI WT and M6PR-CI KO neuroblastoma lines. (F) Glucosylsphingosine measurement in GBA-deficient M6PR-CI WT and M6PR-CI KO neuroblastoma lines. (G) Total GCase activity in GBA-deficient M6PR-CD WT and M6PR-CD KO neuroblastoma lines. (H) Glucosylsphingosine measurement in GBA-deficient M6PR-CD WT and M6PR-CD KO neuroblastoma lines. n=6. Bars represent mean+SEM. Data were analyzed by two-tailed Student's t-test comparing WT vs KO of each receptor for each treatment. *p<0.05; **p<0.01; ***p<0.001, n=6. [Figure 4]hGCase-hBS reverses protein and lipid dysregulation in lysosomes from GBA1 KO H4 cells. (A) Scheme for hGCase-hBS treatment and lysosome isolation from cells. H4 cells expressing lysosome-tag (TMEM192-3HA) are treated with 1 nM hGCase-hBS for 24 h, followed by cell lysis and lysosome isolation using anti-HA coated magnetic beads. (B) Validation of lysosomal enrichment after TMEM192-3HA-based Lyso-IP. Western blot showing enrichment of lysosomes after Lyso-IP, as demonstrated by enrichment of bona fide lysosomal proteins Lamp2, Cathepsin D and GCase in the eluent fraction compared to the input. (C) Rescue of dysregulated proteins upon hGCase-hBS treatment in lysosomes. Graph showing increase or decrease in protein levels in GBA1 KO lysosomes (blue bars) and their rescue upon hGCase-hBS treatment (green bars). (D) Rescue of dysregulated lipids in lysosomes upon hGCase-hBS treatment. Graph showing increased or decreased levels of lipid species in GBA1 KO lysosomes (blue bars) and their rescue upon hGCase-hBS treatment (green bars). [Diagram 5] In vivo GCase-BS proof of concept. (A) PK study in GBA+ / + mice to assess systemic exposure of mGCase-mBS in plasma and brain. n=3 mice per group. (B) Multi-dose PD study in 4L / PS-NA mice to compare equimolar doses of mGCase vs. mGCase-mBS. GlcSph levels were measured in cortex, midbrain and liver. n=6 mice per group. Data are expressed as group mean + / - SEM. Data were analyzed by one-way ANOVA (Dunnett's multiple comparison test) comparing each treatment group to 4L / PS-NA, vehicle. nsp>0.05;****p<0.0001 [Figure 6]Longitudinal effects of GCase-BS in vivo. (A) Single dose PD study in 4L / PS-NA mice to inform about the duration of GlcSph reduction in cortex and midbrain. GlcSph levels rebound between 15-30 days after dosing. n=4-6 mice per group. (B) Longitudinal study in 4L / PS-NA mice with monthly or biweekly dosing frequency. GlcSph levels in cortex and midbrain as efficiency readout. NfL levels in plasma as readout for neurodegeneration. n=10 mice per group. Data are expressed as group mean + / - SEM. Data were analyzed by one-way ANOVA (Dunnett's multiple comparison test) comparing each treatment group with 4L / PS-NA, vehicle. nsp>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] explanation Protein of the Invention [Table 1]

[0045] In GD, currently available treatments, including small molecules such as substrate reduction therapy or enzyme replacement therapy (ERT), fail to target GCase within the CNS compartment.

[0046] To overcome these limitations, we took advantage of the known ability of the transferrin protein to cross the blood-brain barrier (BBB) ​​via binding to the transferrin receptor (TfR), which transports the iron-binding protein transferrin to the brain. 15、16 Furthermore, we reasoned that hijacking this TfR-mediated pathway might also increase lysosomal localization of cargo proteins such as GCase. 17、18With this in mind, a fragment of a transferrin receptor (TfR) antibody was fused to recombinant human or mouse GCase to generate a fusion protein that we named GCase brain shuttle (GCase-BS). We evaluated its efficacy in correcting GBA1-associated molecular alterations in vitro and in vivo. We demonstrated that the GCase-BS TfR binder not only mediates successful transcytosis of GCase across endothelial cells of the BBB, but is also significantly more efficient than conventional ERT using recombinant GCase in delivering the enzyme to the lysosomal compartment and driving hydrolysis of pathologically accumulated lysosomal lipids in multiple neurological models. Our results also reveal GD-associated lysosomal protein and lipid defects that are rapidly corrected within the organelle upon delivery of GCase-BS. Our data provide proof-of-concept support for the use of GCase-BS for the treatment of GBA1-associated neurological dysfunction. Furthermore, this study provides insight into putative GD-associated lysosomal biomarkers downstream of GCase and highlights areas requiring further optimization. Given that TfR targeting is highly effective, it is conceivable that this approach could be extended to multiple LSDs, even in under-targeted peripheral tissues where significant unmet medical need remains.

[0047] The AAV viral particles of the present invention overcome the AAV biodistribution limitations of current CNS AAV gene therapy approaches that use Gcase as a transgene. Currently available AAV capsids targeting the CNS only transduce a portion of neurons resulting in low expression of the transgene in the target tissue. The use of the fusion proteins of the present invention in AAV gene therapy for the CNS allows cross-correction, i.e., the fusion proteins of the present invention are secreted by AAV viral particle-transduced cells, and the secreted fusion proteins of the present invention are taken up by non-transduced cells via TfR. This approach results in more cells with functional GCase and potentially higher efficiency, overcoming the AAV biodistribution limitations of traditional CNS gene therapy approaches that use Gcase as a transgene. Thus, the fusion proteins of the present invention (GCase-TfR binders) can be used as recombinant fusion proteins or expressed directly in vivo from AAV formats.

[0048] Purified GCase-BS molecules are functional with respect to enzymatic activity, stability and TfR binding We designed GCase-BS molecules in which one chain of a human IgG1 Fc portion is fused to the C-terminus of GCase and the other chain of the Fc portion is fused to the N-terminus of an anti-mouse or anti-human TfR-binding Fab. These fusion constructs, called mGCase-mBS or hGCase-hBS, respectively, were designed using knobs-into-hole technology (see 19; Figure 1A), which was used to enable monovalent binding to the TfR.

[0049] We generated various insect cell (S2) derived constructs and analyzed the purified molecules to evaluate their TfR binding and enzymatic properties (Figure 1B-D). Binding to mouse TfR or human TfR was assessed by FACS performed using the appropriate TfR expressing cell lines, with EC50 values ​​of selected fusion constructs of 21 nM for mGCase-mBS or 15 nM for hGCase-hBS, respectively (Figure 1B and C). We compared the enzymatic properties of all molecules described in the manuscript with the conventional ERT molecule imiglucerase (commercially available as Cerezyme). Brain shuttle constructs tend to be more efficient in terms of the ratio of turnover to affinity for resorufin-β-D-glucopyranoside. Naked GCase constructs and TfR non-binders (GCase-NB) are highly similar to imiglucerase (Figure 1C). Furthermore, initial testing of the produced mouse and human GCase-BS constructs by addition to 10% mouse plasma for 15 min maintained both stability and enzymatic activity (Figure 1E).

[0050] TfR-binding modules improve lysosomal targeting and substrate reduction in vitro To investigate the effect of the TfR binding module on cellular uptake and lysosomal efficacy, we used various GCase-deficient cell lines: embryonic null allele Gba- / - mice 20 Immortalized mouse cortical neurons derived from GBA1-deficient human pluripotent stem cell-derived neurons or human neuroblastoma cells (H4 cells) (GBA- / -) 21 , or primary mouse neurons carrying the human GBA1 homozygous mutation (Gba1 D409V / D409V). 22 Both mouse and human cell lines exhibit reduced basal GCase activity and significantly elevated lysosomal glycolipid levels compared to their respective WT cells (Westbroek et al., 2016 and see Figure 2 GBA+ / + vs. GBA- / -).

[0051] In cellular uptake experiments, treatment of immortalized mouse cortical neurons with mGCase-mBS for 2 h resulted in a dose-dependent increase in total GCase activity, with function normalizing at 10–100 nM relative to WT levels. Compared to mGCase or imiglucerase, mGCase-mBS showed a clear increase in cellular uptake, most pronounced at the highest dose (Figure 2A). Consistent also for the human molecule, in GBA1 KO H4 cells, treatment with hGCase-hBS for 2 h increased total GCase activity up to 3-fold over imiglucerase, suggesting that uptake of hGCase-hBS is superior to GCase alone (Figure 2B). Interestingly, uptake of control constructs, mGCase and hGCase fused to TfR non-binders, was less (Figure 2B, hGCase-NB), indicating that the in-house produced GCase constructs had a reduced propensity for cellular uptake and lysosomal retention compared to the commercially available enzyme. This may be explained by the different N-glycosylation patterns on the enzyme surface, which are known to be involved in cellular uptake and intracellular trafficking. 23 .

[0052] We next measured the increase in lysosomal GCase activity in neurons utilizing a novel GCase-specific fluorescence quenching substrate, LysoFQ-GBA (review in peer review). Cellular imaging revealed that lysosomal GCase activity could be restored to WT levels by treating Gba-deficient mouse cortical neurons with mGCase or mGCase-mBS for 2 h. We found that LysoFQ-GBA signal colocalized with the lysosomal probe SiR-lysosome, and quantification of GCase activity showed that mGCase-mBS (EC50=1.5 nM) was approximately 100-fold more effective than mGCase (EC50>100 nM) in restoring lysosomal GCase activity (Figure 2C). In primary neurons derived from Gba1 D409V / D409V mice, GCase activity was approximately 7-fold higher for mGCase-mBS compared to an equimolar dose of mGCase (32 nM, Supplementary Figure 1C). In GBA1 KO H4 cells, treatment with imiglucerase or hGCase-hBS for 2 hours resulted in a significant increase in lysosomal GCase activity. Quantification of the LysoFQ-GBA signal revealed that hGCase-hBS (EC50=10.6nM) was 5-fold more efficient in increasing lysosomal GCase activity compared to imiglucerase (EC50=49nM) (Figure 2D). These data indicate that imiglucerase is less efficiently imported into lysosomes than our hGCase-hBS construct, which actively engages TfR.

[0053] When analyzing the GD pathological lipid glucosylsphingosine (GlcSph) levels in both human and mouse lines 48 hours after treatment with each molecule, we found that in both cell lines, lysosomal hydrolysis was significantly increased when GCase was fused to the TfR-binding moiety. Comparison of IC50 values ​​revealed that mGCase-mBS was more than 100-fold more effective than mGCase alone, and hGCase-hBS was approximately 5-fold more effective than imiglucerase (Figures 2E and 2F). As a control, we showed that hGCase fused to a TfR non-binding module (hGCase-NB) resulted in limited substrate reduction, consistent with its inefficient cellular uptake (Figures 2B and 2F).

[0054] Using immortalized mouse Gba1 KO cortical neurons or GBA1 KO H4 cells, we performed a kinetic study of GlcSph levels whereby cells were incubated with various concentrations of mGCase-mBS and hGCase-hBS for 2 h each, followed by washout, and then monitored the kinetics of GlcSph hydrolysis (Supplementary Figure 1A and B). For both constructs in the respective cell lines, GlcSph levels were halved within 6 h after washing, with maximum efficacy reached by 24 h at all concentrations tested. Interestingly, a dose-response relationship affected the duration of treatment, with short incubations of 100 nM and 10 nM hGCase-hBS resulting in sustained lipid reduction over the 72-h course of the experiment, while 1 nM incubation showed that glycolipid levels began to rebound to those seen in disease states. These data suggest a 2-h incubation is sufficient to degrade substrate levels over an extended period of time, suggesting a mechanism driven by maximum concentrations of GCase in the lysosomes. These data provided the basis for in vivo pharmacokinetic and dynamics (PK / PD) studies.

[0055] In hiPSC-derived macrophages from healthy donors (GBA+ / +) or from PD individuals with the GBA1 genotype N370S / +, treatment with 100 nM hGCase-hBS for 9 days resulted in a significant increase in total GCase activity (+58% in GBA+ / +; +66% in GBA1N370S / +). Analysis of GlcSph levels showed that their levels were significantly elevated in the GBA1 N370S / + line (+88%) and could be substantially reduced upon treatment with hGCase-hBS (55% reduction at 100 nM, 29% reduction at 10 nM). These data suggest that the construct is functional in macrophages and normalizes enzyme activity and GlcSph lipids in cell models with GD / PD pathological mutations.

[0056] Human pluripotent stem cell-derived GBA1 KO dopaminergic neurons 24 Thus, treatment with 10 nM hGCase-hBS efficiently normalized GlcSph levels, suggesting that hGCase-hBS is effective at low concentrations in human midbrain neurons associated with PD and GD.

[0057] Mode of GCase-BS lysosomal action in vitro It is well established that TfR-binding agents transport therapeutic molecules across the BBB 18、25、26However, it remains unclear how the BS module of GCase-BS serves to promote CNS cell uptake and lysosomal targeting of GCase. Inspired by the finding that the BS module increases both the exposure to lysosomes and the efficacy of hydrolysis of GlcSph within lysosomes, we aimed to elucidate the underlying mechanism in more detail. To this end, we compared the ability of four constructs: 1) GCase attached to a TfR-binding molecule (hGCase-hBS), 2) GCase attached to a TfR non-binder (hGCase-NB), 3) antibody cargo (NB-hBS) and 4) BS (hBS), and a control BS (NB) moiety only, to target lysosomes. GCase-deficient H4 cells were incubated with the molecule for 2 h and the localization of both the brain shuttle moiety and the enzyme was monitored. We found that cellular uptake and exposure to lysosomes of hGCase-NB was negligible, which we assessed based on little or no IgG or hGCase cellular immunoreactivity colocalized with LAMP1 (Figures 3A and 3B). This observation was consistent with previous findings that hGCase-NB alone resulted in very limited cellular uptake and reduced lysosomal lipids (Figures 2B and 2F). We also observed that 2-hour treatment with hGCase-hBS colocalized both GCase (up to 50% colocalized GCase spots at 100 nM) and hBS (up to 25% colocalized hIgG spots at 100 nM) with LAMP1. We observed that NB-hBS or hBS alone showed significantly less colocalization (Figures 3A and 3B), suggesting that the combination of both the BS module and its GCase cargo is required for efficient lysosomal targeting.

[0058] To determine which receptor is required by hGCase-hBS for both cellular uptake and lysosomal targeting / efficacy, we generated several double KO H4 strains lacking either GCase and TfR or cation-dependent M6PR (M6PR-CD) or cation-independent M6PR (M6PR-CI) to obtain GBA / M6PR-CD KO, GBA / M6PR-CI KO and GBA / TfR KO cells. After 2 hours of treatment with imiglucerase, hGCase-NB or hGCase-hBS, we observed that the absence of TfR resulted in both a marked reduction in cellular uptake of the construct and a marked impairment in the ability of the construct to reduce GlcSph. In contrast, for imiglucerase, cellular uptake and efficacy remained unaffected (Figure 3C and Figure 3D). However, using M6PR-CI KO, we found that both the uptake of the enzyme and its ability to reduce lysosomal glycolipids were greatly affected for imiglucerase, whereas the activity of hGCase-hBS was not affected (Figures 3E and 3F). Interestingly, we found that M6PR-CD played no role in either cellular uptake or lysosomal lipid reduction for either molecule (Figures 3G and 3H).

[0059] Taken together, our data suggest that, as expected, imiglucerase uses M6PR-CI for cellular and lysosomal uptake, whereas the GCase-BS construct primarily accesses lysosomes by engagement and sorting via its interaction with TfR.

[0060] GCase-BS corrects the lysosomal phenotype Having demonstrated the efficacy of the brain shuttle in delivering GCase to lysosomes in neuronal cell lines, we sought to investigate the consequences of restoring lysosomal GCase on the molecular structure of lysosomes. As treatment of GBA KO cells with 1 nM hGCase-hBS was sufficient to normalize GlcSph levels without saturating the system, we used this condition to better understand lysosome-specific changes in proteins and lipids. To this end, we established an experimental paradigm to specifically enrich and profile lysosomes for perturbations of the proteome and lipidome upon hGCase treatment (Figure 4A). We used a method to purify lysosomes from cells that relies on stable expression of the so-called lysosomal tag TMEM192-3XHA 27. Expression of the lysosomal-tag facing the cytosolic side allows for rapid and efficient immunoprecipitation of lysosomes using anti-HA antibodies after cell lysis (Figure 4A). After treating cells with hGCase-hBS, we purified lysosomes in this way and confirmed their intactness by examining the presence of the lysosomal membrane protein Lamp2 and the soluble lysosomal luminal proteins Cathepsin D and GCase (Figure 4B, eluent fraction). The isolated lysosomes as well as the whole cell extract were subjected to global protein profiling using HRM™ ID / ID+ mass spectrometry. Proteomic analysis showing enrichment of authentic lysosomal proteins in the isolated lysosomal fraction compared to the whole cell extract further validated the isolation protocol delivering intact lysosomes (Supplementary Figure 3). The list of authentic lysosomal proteins includes proteins shown to be of lysosomal origin by comparative proteomic analysis of lysosomes from mammalian cells 28、29Principal component analysis (PCA) of the proteomic dataset showed a clear separation between the lysosomal and whole cell extract groups, as well as between the WT and GBA KO groups. Several proteins were significantly up- or downregulated in lysosomal and whole cell extracts from H4 GBA KO cells compared to those from H4 WT cells (Figure 4C and Supplementary Figure 5). Treatment of H4 GBA KO cells with 1 nM hGCase-hBS was sufficient to rebalance the altered protein levels in both lysosomes (Figure 4C) and whole cell extracts. One of the proteins significantly increased in GCase-deficient lysosomes is the pro-inflammatory mediator S100A9 (log2FC-GBA KO lysosomes vs. WT lysosomes: 1.77). Interestingly, S100A9 has been shown to colocalize and coaggregate with alpha-synuclein in Lewy body forms of PD patients, and in vitro studies suggest that S100A9 may alter the aggregation kinetics of alpha-synuclein. 30、31 Treatment with hGCase-hBS was able to efficiently reverse the increase in S100A9 levels observed in GCase-deficient lysosomes (log2FC-GBA KO-hGCase lysosomes vs. GBA KO lysosomes: -2.49). Another interesting protein significantly increased in GCase-deficient lysosomes and efficiently reverted by hGCase-hBS treatment is estrogen-related receptor alpha (ESRRA) (log2FC-GBA KO lysosomes vs. WT lysosomes: 3.27; log2FC-GBA KO-hGCase lysosomes vs. GBA KO lysosomes: -3.17). ESRRA has been shown to increase cellular expression of monoamine oxidase (MOA), the mitochondrial enzyme responsible for the oxidation of dopamine. Furthermore, parkin has been shown to negatively regulate this process by ubiquitination and degradation of ESRRA. 32 .

[0061] Using the same experimental paradigm used in the proteomic study, we performed a mass spectrometry-based shotgun lipidomic analysis of lysosomal and whole cell extracts to evaluate the impact of hGCase-hBS on lipid profiles in GCase-deficient H4 cells. Many lipid species belonging to the hexosylceramide (HexCer) family were increased in lysosomal and whole cell extracts lacking GCase and efficiently returned to basal levels with 1 nM hGCase-hBS (Figure 4D). Our data also confirm that in addition to GlcSph, GCase catalyzes the degradation of several species of HexCer that differ in chain length, double bonds and hydroxylation. We also found that the levels of lipid species belonging to phosphatidylcholine (PC), phosphatidylglycerol (PG) and phosphatidylserine (PS) were significantly altered in GCase-deficient cells (Figure 4D). Notably, several of these altered lipid species were subsequently corrected to basal levels upon treatment with hGCase-hBS (Figure 4D). Collectively, our data suggest that GCase deficiency in cells leads to global lipid alterations that can be corrected in a rapid time frame following incubation with hGCase-hBS.

[0062] In vivo GCase-BS proof of concept Next, we performed a single dose study using C57BL / 6 mice to analyze mGCase-mBS levels, measuring both the BS portion of the construct (total IgG) by IgG immunoassay and GCase enzymatic activity using a chemical substrate. In this way, we were able to evaluate the pharmacokinetics (PK) and stability of the molecule in vivo. The mGCase-mBS construct shows a high systemic clearance (>30 ml / h / kg). Initially, the plasma levels measured by both assays were similar (5 min after injection), but over the entire observation period, the exposure in terms of area under the curve (AUC) was approximately 4-fold lower when measuring the enzymatic activity compared to the exposure measured using the IgG immunoassay (Figure 5A). This suggests that the stability of the GCase domain was affected in the blood over time. As uptake into the brain is the rate-limiting step, the assessment of brain exposure by measuring total IgG levels from brain lysates showed a slightly lower but parallel pharmacokinetic profile (Cmax for total IgG in brain at 24 h: 2.1 nM). No active mGCase could be detected in plasma after 24 h, possibly due to lack of sensitivity of the GCase enzyme activity assay (LOQ=0.4 nM).

[0063] To demonstrate proof-of-concept of the GCase-BS construct in vivo, we first performed a multi-dose (4 doses of 2.5 mg / kg) study in a mouse model of GD named 4L / PS-NA. These mice have a homozygous Gba1 mutation (Gba V394L / V394L) and prosaposin KO (Psap- / -). The mice display a neuronal phenotype similar to that of, for example, GD2 or GD3 patients, and show reduced GCase activity and strong accumulation of GlcCer and GlcSph in the lysosomal compartment. 33、34Assuming that pharmacokinetics in the brain in these mice also runs in parallel with the blood profile, the rationale for the dosing regimen was to generate a high enough brain exposure to allow target engagement and thus induce clear pharmacodynamic effects on relevant markers. We found that at baseline, 4L / PS-NA mice showed an increase in GlcSph levels to 4-fold (cortex), 3-fold (midbrain) or even 13-fold (liver) compared to control littermates. 24 hours after the last dose of mGCase-mBS in 4L / PS-NA mice, lipid levels in both the cortex and midbrain were reduced by about 72% (Figure 5B). Unconjugated mGCase alone was not effective in reducing lipids in multiple brain regions, suggesting that the BS module is the key to reducing brain lysosomal GlcSph (Figure 5B). However, in the liver, both mGCase and mGCase-mBS led to about 60% normalization of the substrate, suggesting that they are similarly potent in the liver, which was unexpected based on the observation that TFR is more efficient (Figure 5B). This unexpected observation may be explained by the differential expression pattern of M6PR and TfR in this tissue compared to brain tissue, which contains almost no TFR in hepatocytes. Similar results were obtained in mice with only the Gba1 mutation (Gba V394L / V394L). In summary, these results suggest that the brain shuttle module not only increases lipid reduction capacity but also promotes crossing of the BBB into the brain parenchyma in the two Gba1 mouse models.

[0064] Subsequent single-dose dose-response experiments were performed with doses ranging from 0.2 mg / kg to 2.5 mg / kg, and results suggested that doses below 2.5 mg / kg were not sufficient to significantly reduce brain lysosomal lipid levels. Therefore, to gain more insight into the appropriate dosing frequency and the course of pathological lipid rebound after the last dose, we injected a single dose of either 2.5 mg / kg or 10 mg / kg and analyzed tissues at various time points after the last dose in 4L / PS-NA mice (4-6 mice per group). GlcSph analysis revealed that both doses similarly resulted in a ∼50% reduction in substrate relative to control animals at 5 days post-injection. The kinetics of change in GlcSph levels revealed that 15 days post-injection, GlcSph levels were still significantly lower in both the cortex and midbrain, and it took up to 45 days to return to the levels of GlcSph seen in untreated animals. These results suggest that lipid reduction following treatment is sustained and that a biweekly or monthly dosing frequency may be sufficient to reach beneficial effects (Figure 6A).

[0065] To test whether this is true, we performed a multiple-dose chronic study in which 4L / PS-NA mice were injected monthly or biweekly with 2.5 mg / kg mGCase-mBS for 3 months starting at 1 month of age. In this study, we monitored GlcSph levels in the brain and plasma NFL levels (a biomarker of neurodegeneration previously reported to be elevated in this model) as a biomarker of neurodegeneration. 33Two weeks after the last injection, there was a reduction of about 18% and about 30% in substrate levels in both the cortex and midbrain, respectively (Figure 6B). We did not observe any significant differences between the biweekly and monthly dosing regimens. Of note, NFL levels were elevated 35-fold in 4LPS-NA mouse plasma compared to controls. Using both dosing regimens, we observed a significant reduction to nearly 30% of untreated 4LPS-NA. These results suggest that both biweekly or monthly dosing are adequate to lower lipids sufficiently to alter the course of neurodegeneration in an aggressive GD model (Figure 6B).

[0066] Consideration The classical view of lysosomes is as the final catabolic station that relieves cells of waste products. 35 This view has recently been expanded by the discovery of new roles for lysosomes in nutrient sensing, transcriptional regulation, and metabolic homeostasis. 35 At the intracellular level, LSDs are manifested in the abnormal intralysosomal accumulation of metabolic products resulting from defects in one or more catabolic pathways caused by genetic defects that result in reduced levels of lysosomal enzymes. 36 Although the clinical symptoms of LSD vary widely, neurological symptoms are a common feature. 37 Restoring levels of the missing enzyme is a highly effective treatment and is the standard of care in many different types of LSD. However, the recombinant enzyme used in ERT lacks the ability to cross the BBB. This results in no or insufficient exposure of the therapeutic enzyme to the brain and subsequent failure to reverse the patient's neurological complications.

[0067] GD (GD2 and GD3) and the neuropathic forms of GBA-PD are devastating neurological disorders that manifest as a consequence of GCase lysosomal dysfunction leading to neurodegeneration. Compelling preclinical and clinical data exist suggesting that increasing GCase activity in the brain could reverse the underlying lysosomal dysfunction and thus have a significant impact on the disease course. 38Taken together, our data suggest that fusion proteins containing GCase and TfR binders represent a promising therapeutic approach for GBA1-associated neurological diseases. Current ERT of GCase, an effective treatment for GD1, has various limitations that can be addressed to optimize efficacy versus CNS potency, as reflected in markers including pathological lysosomal lipid reduction, rapid clearance from blood, and enzyme stability after infusion. Thus, the GCase-BS construct breaks new ground by offering significant potential advantages. We show that it not only crosses the BBB endothelium but also results in 5- to 100-fold more efficient glycolipid reduction in multiple relevant human cell lines (Figure 2).

[0068] Recently, it has been shown in both preclinical and human clinical trials that the lysosomal enzyme iduronate 2-sulfatase (IDS) bound to a TfR binder can cross the BBB with demonstration of CNS target engagement (e.g., CSF / NFL). 39、40 This effort culminated in the approval of JR-141, an IDS TfR binder, for mucopolysaccharidosis (MPS) type I, the central disease manifestation in Japan. 41 Recent studies have also highlighted that this approach extends to other lysosomal proteins, including progranulin. The concept of TfR binding to cross the BBB to treat MPS2 and neuronal ceroid lipofuscinosis (NCL) has been validated in humans. 42、43 Here, we extend these findings for GBA1-related neurodegenerative diseases.

[0069] Soluble and membrane-bound lysosomal proteins have sorting signals that are recognized by sorting receptors for their appropriate delivery to the endolysosomal system via various trafficking pathways. GCase, as a lysosomal enzyme, follows a rather unique route to the lysosome that involves the lysosomal integral membrane protein 2 (LIMP2) receptor. 44Sorting signals for soluble lysosomal proteins can be either folded polypeptide sequences displayed on the protein surface or specific glycan modifications. Modification with mannose 6-phosphate (M6P) is a well-characterized sorting signal, and in the case of GCase ERT, the glycans are adjusted to make the "mechanism of action" dependent on this pathway. When tagged with M6P residues, the enzyme is sent to the lysosome by interaction with the cation-independent mannose 6-phosphate receptor (M6PR-CI, also IGFR2). M6PR-CI is localized not only in the trans-Golgi network and endosomes, but also in the plasma membrane, so 45、46 In this study, exogenously delivered GCase (ERT) can be retrieved by M6PR-CI and sorted into the endolysosomal system and subsequently to lysosomes. This mechanism represents the basis of effective ERT and is used by commercially available forms of GCase that contain the enzyme imiglucerase.

[0070] In the case of GCase-BS, we demonstrated that uptake, lysosomal exposure and hydrolysis of pathological GCase-targeted lipids is much more efficient and potent than the enzyme alone. We show that this "mechanism of action" depends only on TfR and not on the traditional receptor M6PR for ERT. Notably, a GCase construct with an inactive TfR binder (hGCase-NB) showed no detectable uptake or substrate reduction, indicating that this construct cannot effectively use the M6P pathway via M6PR-CI sorting (Figure 3). This suggests that hGCase-NB either lacks the glycan modifications required to engage M6PR-CI or that brain shuttles prevent efficient uptake. However, when an active TfR binder is used as in the GCase-BS construct, strong cellular uptake and strong substrate reduction are seen. Thus, in the neuronal cells used in this study, TfR provides an efficient route to the lysosome. Importantly, at the BBB, this TfR sorting pathway is very likely different. Herein, brain shuttle constructs are transported across the BBB to the brain parenchyma if their engagement with TfR has certain characteristics, including a monovalent binding mode that appears to prevent lysosomal sorting in endothelial cells at the BBB. Collectively, the data presented in this study indicate that the TfR pathway can provide both productive transport across cells at the BBB and subsequent enhanced uptake and efficacy in hydrolysis of lysosomal glycolipids in neuronal cells in the brain. It is also important to understand that the cargo associated with the BS in the fusion construct can affect the transport of the construct. For example, if the cargo is a high affinity antibody to a specific target in the brain, the BS construct will likely be directed to this target for stronger binding compared to binding to the TfR. Thus, it remains to be determined whether other lysosomal enzymes coupled to TfR binders share the characteristics seen here for GCase-BS, including increased uptake and efficacy.

[0071] Upon systemic administration of mGCase-mBS, rapid clearance of the construct from the blood is observed (Figure 4A). Notably, GCase enzyme activity appears to decline in the blood much faster than the shuttle domain of the construct. Only about 0.1% of the injected enzyme activity is detectable in the circulation after 24 hours. This suggests that the GCase enzyme is much less stable in the blood in vivo than the brain shuttle, likely facilitating clearance of the construct from the peripheral compartment. This observation highlights current limitations that could be further optimized, including clearance and enzyme stability. As it was clear that the molecule lost stability over time in the blood, attempts could be made to stabilize GCase. For example, it has been reported that GCase mutants could be improved in stability. 47、48 The short half-life and stability of the GCase-BS construct likely limit its efficacy. The combination of pharmacological chaperones with ERT has shown potential to improve the bioavailability of another recombinant human GCase enzyme in preclinical studies and clinical trials. 49~52 This strategy can be applied to GCase-BS using known GCase chaperones such as isofagomine. Such an approach can stabilize GCase-BS allowing more active enzyme to cross the BBB. Uptake of the molecule depends only on TfR binding and is therefore independent of terminal mannose residues, suggesting that tailoring the glycans on the construct can increase stability and bioavailability. For example, the negative charge of sialic acid is thought to prevent aggregation by creating repulsion between therapeutic molecules and preventing renal filtration. 53 Thus, polysialylation of therapeutic agents has been shown to significantly increase their half-life. 54、55 Future experiments will be needed to test these concepts.

[0072] Mouse models of Gba1-related PD have limitations that affect their usefulness for studying disease biology. Such models, for example, have little to no lipid accumulation in the brain. 56 Therefore, we utilized the 4LPS / NA model, which has a homozygous knock-in of Gba V394L / V394L and only one allele of psap. This mouse model has long-term accumulation of brain lipids and was ideal for the purpose of monitoring pharmacodynamic effects on lipids such as GlcSph. We showed that a single dose of mGCase-mBS reduces GlcSph levels by nearly 50% compared to WT levels, but four doses spaced 24 hours apart (loading phase) result in a reduction of about 70% compared to WT levels. These results mechanistically imply that the in vivo effects in the brain are driven in part by the maximum concentration in blood (Cmax), which is supported by in vitro studies showing that lysosomal hydrolysis is fast, but that the ability of TfR at the BBB to shuttle mGCase-mBS may become saturated. This view is supported by observations showing that doses of 1-10 mg / kg result in a limited dose-response relationship.

[0073] Within the liver, both the free GCase enzyme and the mGCase-mBS construct were highly active, validating that GCase ERT is functional and active in vivo outside the brain, but is unable to penetrate the CNS. The similar efficacy of both mGCase and mGCase-mBS in the liver could be explained by the different expression levels of M6PR and TfR in this tissue compared to brain cells. Another notable observation is the marked and sustained drop in GlcSph levels in the brain after the end of treatment. In both the cortex and midbrain, a significant decrease in GlcSph was observed up to 15 days after treatment, with a clear trend even up to 45 days (Figure 4C). Recently, it has been shown that NF-L levels in CSF and plasma in this 4L / PS-NA GD mouse model were 9-fold higher in plasma and 70-fold higher in CSF compared to wild-type controls. 57In this study, a similar increase in plasma NF-L was observed (Figure 4D). Treatment with the mGCase-mBS construct for 12 weeks, using a dosing frequency (biweekly / monthly) that would be acceptable for delivery of human therapy to treat GD or GBA-PD, was able to significantly reduce these abnormal NF-L levels. This measurable therapeutic effect likely occurs within the CNS, as NF-L is found in large myelinated axons of neurons. The roughly 30% reduction in NF-L to WT levels is striking, as this model has a component of neurodegeneration independent of GCase, driven by PSAP haploinsufficiency, where PSAP affects other lysosomal functions, such as progranulin metabolism. 58 .

[0074] CSF GlcSph is a putative translational biomarker for both GD and GBA-PD downstream of elevated GCase in the brain. However, as highlighted by the recent venglustat clinical trial, reduction of these important pathological lipids is not sufficient to determine whether GBA1-dependent lysosomal homeostasis has been restored. 59、60 To that end, we monitored protein and lipid changes in both whole cell lysates and lysosomes rapidly purified from GD cell models. We identified both key proteins and lipids that are abnormally regulated in GD cells and rapidly reverted after addition of hGCase-hBS. Future studies can address the role of these molecular changes in GBA1-associated neurodegeneration and may ultimately serve as downstream proximal GBA1 pathway lysosomal biomarkers of target engagement. Key translational biomarkers may require rapid lysosomal purification of peripheral cells to demonstrate functionality in the lysosomal compartment. 35 .

[0075] In summary, this study suggests that GCase-TfR binder fusion proteins can correct lysosomal defects and thus represent an attractive therapeutic avenue for GBA1-associated neurodegeneration. We further suggest opportunities for future optimization of such constructs and provide the basis for potential biomarkers that can be used to examine the efficacy of lysosomal-targeted therapeutics in a translational context.

[0076] method Reagents and antibodies Unless otherwise stated, chemicals were purchased from Sigma Aldrich.

[0077] Antibodies used to perform immune-based experiments were: rb mAb~hGCase (Abcam, #ab128879), rb mAb~TFRC (Abcam, #ab214039), rb mAb~M6PR cation-independent (Abcam, #ab124767), rb mAb~M6PR cation-dependent (Abcam, #ab134153), HRP-conjugated rb pAb~GAPDH (Abcam, #ab9385), ms mAb~Lamp2 (Thermo Fisher. #MA1-205), rb mAb~cathepsin D (Abcam, #ab75852).

[0078] cell culture Human glioma cells (H4) were maintained in DMEM / F-12 (#11039-021) supplemented with 10% fetal bovine serum (#A31605-01) and penicillin (100 U / ml), streptomycin (100 μg / ml) (ThermoFisher).

[0079] Human neurons were cultured from neural stem cells (NSCs) for 6 weeks in differentiation medium (DMEM / F-12 with GlutaMax (#31331093) and supplemented with 1x B27 (#12587010), 1x N2 (#17502048), 0.1% (v / v) β-mercaptoethanol (#31350010), penicillin (100U / ml), streptomycin (100μg / ml), laminin (1 / 500) and cytokines: 20ng / ml BDNF (Peprotech#450-02), 10ng / ml GDNF (Peprotech#450-10), 100μM ascorbic acid 2-phosphate (Sigma#A8960) and 500μM β-mercaptoethanol (Sigma#A8960). Differentiation was performed in Neurobasal Medium (#21103049) supplemented with cAMP (Sigma #D0627-5X1G).

[0080] Mouse immortalized primary neurons were maintained in neurobasal medium supplemented with 1×B27, 1×GlutaMax, penicillin (100 U / ml), streptomycin (100 μg / ml) and laminin (1 / 500).

[0081] All cells were kept at 37°C in a humidified 5% CO2 atmosphere.

[0082] animal For in vivo studies, we used the 4L / PS-NA mouse model, which resembles some of the neuropathic phenotypes of GD.

[0083] These animals contain a V394L mutation in the GCase locus and a prosaposin gene knockout (Sun et al., 2005). Control littermates (WT for the prosaposin gene) were used as baseline.

[0084] Breeding and research were carried out at QPS Austria in accordance with the respective animal handling regulations.

[0085] Generation of H4 KO cell line CRISPR / Cas9 gene editing was performed to generate several KO H4 cell lines: Briefly, H4 GBA KO cells were seeded in 6-well plates at 1E5 cells / well and transfected with RNP at a ratio of 1.8:1 (sgRNA:Cas9 nuclease) by lipofection. Lipofection reagent was purchased from Thermo Fisher Scientific (Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent #CMAX00015). Media was replaced with complete growth media after 18 hours and cells were subjected to limiting dilution to obtain monoclonal cell populations. Protein loss was confirmed by Western blot.

[0086] Synthetic sgRNA was purchased from Synthego: 1) TfRC: Guide #1: G*U*G*AUCGUCUUUUUCUUGAU (SEQ ID NO: 8) Guide #2: A*A*A*UGCUGACAAUAACACAA (SEQ ID NO: 9) Guide #3: A*G*A*UGGCGAUAACAGUCAUG (SEQ ID NO: 10) 2) M6PR-CD: Guide #1: A*A*U*CAACAAAAGUAAUGGGA (SEQ ID NO: 11) Guide #2: U*U*C*AGGGUGUGCCGGGAAGC (SEQ ID NO: 12) Guide #3: U*A*C*AGCUUUGAGAGCACUGU (SEQ ID NO: 13) 3) M6PR-CI: Guide #1: U*U*G*AAUUGUGCAGGUAACGA (SEQ ID NO: 14) Guide #2: C*G*U*GUCCCAUGUGAAGAAGU (SEQ ID NO: 15) Guide #3: C*G*U*CGGUGGCACCGCAGAGG (SEQ ID NO: 16)

[0087] The absence of the respective proteins was determined by Western blot analysis. Briefly, cells were lysed in RIPA buffer supplemented with protease inhibitors (Roche, Cat. No.: 11 873 580 001) and 10 μg of total protein was subjected to SDS-PAGE (Invitrogen NuPAGE system). Proteins were transferred onto nitrocellulose membranes using semi-dry transfer for 6 min at 23 V, which were blocked in 5% milk / TBST (Tris-buffered saline + 0.05% Tween) and subsequently incubated with primary antibodies at 1 / 1000 overnight at 4°C. After three washes in TBST, HRP-labeled secondary antibodies were incubated at 1 / 10000 in 5% milk / TBST for 1.5 h at room temperature. Proteins were detected using the SuperSignal West Dura Extended Duration Substrate Kit (Thermo).

[0088] Expression and purification of recombinant human and mouse GCase(-BS) constructs Recombinant hGCase and mGCase, as well as hGCase-hBS and hGCase-NB, were expressed in Schneider S2 cells (a Drosophila cell line) using the expression vector pExpreS2_1-A. Constructs of hGCase and mGCase were designed to be expressed with a C-terminal His8 tag containing a glycine-serine (GS) linker and a sortase recognition site (Sor). hGCase-hBS was designed by fusing one chain of a human IgG1 Fc moiety lacking Fcγ receptors to the C-terminus of human glucocerebrosidase and the other chain to an anti-human TfR-binding Fab at the N-terminus using knob-into-hole technology. mGCase-mBS was expressed in two parts (mGCase-Sor and anti-mouse TfR-binding Fab) and then coupled by sortase-mediated site-specific conjugation.

[0089] To purify mGCase or hGCase, the cell supernatant was filtered and passed through a HiTrap Con A 4B column (GE Healthcare) using HiTrap Con A buffer (20 mM Tris / HCl, pH 7.4, containing 0.5 M NaCl, 1 mM MnCl2, 1 mM CaCl2, 0.02% (v / v) NaN3 and 0.5 M methyl α-D-mannopyranoside for elution). The eluted target protein was further purified on a HisTrap HP column (GE Healthcare) using HisTrap buffer (50 mM HEPES at pH 7.6 with 0.5 M NaCl, 0.02% NaN3 and 0.5 M imidazole for elution) followed by hydrophobic interaction chromatography (HIC) on a Toyopearl Butyl-M 650 HIC column (Tosoh Bioscience) using HIC buffer (20 mM MES at pH 5.5 with 0.5 M KCl, 0.02% NaN3) for binding and 80% (v / v) ethylene glycol for elution.

[0090] To purify hGCase-hBS or hGCase-NB, the cell supernatant was clarified by filtration and loaded onto a HiTrap Con A 4B column (GE Healthcare) as a first purification step. HiTrap Con A buffer (20 mM Tris / HCl at pH 7.4 with 0.5 M NaCl, 1 mM MnCl2, 1 mM CaCl2, 0.02% (v / v) NaN3 and 0.5 M methyl α-D-mannopyranoside for elution) was used. The eluted target protein was further purified by a Capture Select KappaXL column (GE Healthcare) column using 25 mM Tris / HCl (pH 7.0), 25 mM NaCl, 5% (v / v) glycerol, 0.02% NaN3 as binding buffer. An additional wash step with binding buffer and 1% (w / v) CHAPS was included to remove endotoxins. The target protein was eluted from the column with 20 mM citric acid, 0.1 M glycine, 5% (v / v) glycerol and 0.02% NaN3 at pH 3.5. The pH was adjusted to pH 6.0 immediately after protein elution.

[0091] Finally, all purified constructs were dialyzed against a weakly acidic solution (20 mM histidine, 140 mM NaCl, pH 6.0) for further experiments.

[0092] Biochemical characterization of GCase-BS The fluorogenic substrate resorufin-β-D-glucopyranoside (res-β-glc; Sigma-Aldrich) was used to determine the enzymatic activity of the various GCase(-BS) constructs. GCase cleaves this substrate into glucose and resorufin. Product formation of resorufin was measured over time in assay buffer containing 50 mM citric acid pH 6.0, 50 mM KPi, 110 mM KCl, 10 mM NaCl, 1 mM MgCl2 and 1% DMSO at 37°C with excitation at a wavelength of λ = 535 nm and emission at λ = 595 nm. Prior to kinetic measurements, all assay components were preheated to the assay temperature and the fluorogenic substrate was kept in the dark. The reaction was initiated by adding GCase to a final concentration of 25 nM. Raw data from a fluorescence plate reader (Spectramax i3, Molecular Devices) was aggregated in Microsoft Excel and analyzed using GraphPad Prism 8.4.2.

[0093] FACS-based assessment of TfR binding Binding of GCase-BS fusions was tested using mouse-TfR expressing cell line BA / F3 (DSMZ, ACC-300) or human-TfR expressing CHO cells (ATCC, CCL-61 transfected to stably overexpress human TfR). Briefly, suspended cells were harvested, counted, checked for viability, and resuspended at 2 million cells per ml in FACS buffer (PBS containing 0.1% BSA). 100 μl of cell suspension (containing 200,000 cells) was incubated with increasing concentrations of GCase fusions (10 pM to 1 μM) for 1 h at 4 °C in round-bottom 96-well plates. Cells were then washed twice with cold PBS / 5% FBS and re-incubated with labeled secondary antibody (PE conjugated, goat anti-hu IgG (Fc-spec.) from Jackson ImmunoResearch #109-116-170 at a dilution of 1:100 for 30 minutes at 4° C. in the dark and washed twice with cold PBS / 5% FBS. Fluorescence was analyzed by FACS using a BD FACSCanto™ II (Software FACS Diva and FlowJo 10.6.2). Binding curves and EC50 values ​​were obtained using GraphPadPrism 7.

[0094] Liquid chromatography-mass spectrometry of GlcSph Analytes and internal standards were purchased from Avanti Polar Lipids: D-glucosyl-β-1-1'-D-erythro-sphingosine (no. 860535) and D-glucosyl-β-1-1'-D-erythro-sphingosine-d5 (no. 860636) as internal standard 1; D-galactosyl-β-1-1'-D-erythro-sphingosine (no. 860537) and D-galactosyl-β-1-1'-D-erythro-sphingosine-d5 (no. 860637) as internal standard 2. For chromatography, HPLC grade solvents as well as Millipore water were used. Acetonitrile (LiChrosolv number 1.00030) and methanol (LiChrosolv number 1.06007) were obtained from Supelco (Merck) and ammonium acetate for mass spectrometry was purchased by Sigma-Aldrich (number 73594).

[0095] The analysis was performed on an LC-MS-MS system consisting of a Waters Xevo-TQ-S mass spectrometer connected to a complete Waters Acquity I-class UPLC system equipped with a flow-through needle sample manager, using a mixture of acetonitrile / methanol / water 40 / 40 / 20 (v / v / v) as the wash solvent. The autosampler temperature was set at 10 °C.

[0096] Stock solutions for analytes and internal standards were prepared at a concentration of 1 mM in DMSO and kept at -20 °C. For further spiked solutions, acetonitrile / water 9 / 1 (v / v) was used as the solvent. Calibration solutions were prepared by serial dilutions in acetonitrile / water 9 / 1 (v / v) containing 2% DMSO. The concentration range was C1 = 10 μM to C9 = 0.0039 μM. To avoid inhibitory effects coming from the biological matrix, the final calibration samples were made in pooled tissue homogenates and were prepared in exactly the same way as the brain and liver samples.

[0097] Frozen tissue was weighed into 7 ml hard tissue homogenizing vials pre-filled with ceramic beads (Bertin, catalogue number 03961-1-002.2 (CK28) supplied by LabForce AG, Switzerland, or catalogue number 19-628 from Omni International) and homogenized in distilled water to give a final concentration of 100 mg tissue / ml. Samples, QC and calibration samples were cleared by protein precipitation with methanol containing an internal standard.

[0098] After centrifugation, the supernatant was evaporated to dryness, reconstituted in acetonitrile / water 90 / 10 (v / v) containing 1% DMSO, and analyzed by LC-MS / MS. For calibration, a linear regression function with 1 / y weighting excluding 0 was used. The calibration range was x+39 nM (C9) to x+1 μM (C1), where x is the endogenous substrate level in pooled tissue homogenates. Absolute concentrations were calculated by dividing the peak area ratio analyte / internal standard by the slope of the calibration curve.

[0099] Samples were analyzed on a BEH glycanamide column (100 x 2.1 mm, 1.7 μm particle size, purchased from Waters, Switzerland) at a flow rate of 0.25 ml / min and an oven temperature of 30°C. Eluent A consisted of 100 mM ammonium acetate and acetonitrile was used as eluent B. Sugar-specific separation was achieved by isocratic elution with 90% B followed by a wash step with 10% B and column reconditioning. The total analysis time was 12 min.

[0100] The Xevo TQ-S instrument was operated in positive ion electrospray mode with both quadrupoles tuned to unit mass resolution using nitrogen as the nebulizer and desolvation gas. The nebulizer gas flow rate was set at 150 l / h, the desolvation gas flow rate at 800 l / h, and the temperature at 500 °C. Argon was used as the collision gas at a flow rate of 0.15 ml / min. Analytes and internal standards were detected by multiple reaction monitoring mode (MRM) following the transitions m / z 462.3 to 282.3 and m / z 467.3 > 287.3 with a cone voltage of 30 V and collision energy of 18 V.

[0101] Cellular assays to assess uptake, lysosomal activity and potency of GCase-BS A GBA-deficient cell line was used to determine the uptake, lysosomal activity and potency of the GCase-BS molecule.

[0102] To assess cellular uptake, the activity of GCase was determined from whole cell lysates. Cells were seeded in 96-well plates at 5E4 cells / well and maintained at 37 °C, 5% CO2, and 85% humidity for 16–18 h. Cells were then treated with various concentrations of GCase-BS molecules for 2 h. Afterwards, cells were washed once with PBS and lysed in 30 μl of lysis buffer (0.05 M citric acid, 0.05 M KH2PO4, 0.05 M K2HPO4, 0.11 M KCl, 0.01 M NaCl, 0.001 M MgCl2, pH 6.0, containing 0.1% (v / v) TritonX-100, supplemented with freshly added protease inhibitors). 10 μl of cell lysate was mixed with 10 μl of 10 mM resorufin-β-glucopyranoside and baseline fluorescence was immediately measured at t0. Accumulation of fluorescent product (=resorufin) was measured after 2 h incubation at 37° C. (λex=535 nm and λem=595 nm) and indicated GCase activity. Data were normalized to WT cells.

[0103] To assess lysosomal activity, we used a fluorescent quenched GCase substrate (FQ-7), which only emits light when GCase is hydrolyzing it (thus releasing the quencher). Cells were seeded in 96-well plates at 1E4 cells / well and maintained at 37 °C, 5% CO2, and 85% humidity for 16-18 h. Cells were then treated with various concentrations of GCase-BS molecules for 2 h. Afterwards, cells were washed with PBS and a mixture of FQ-7 and SiR lysokit was added at 10 μM and 5 μM, respectively, for 1 h. Cells were washed once more with PBS and Hoechst (final concentration 2 μM) was added for 3 min. Cells were imaged live using Opera Phenix Plus (Perkin Elmer).

[0104] To evaluate the efficacy of GCase-BS molecules, cells were seeded in 96-well plates at 2E4 cells / well and maintained at 37 °C, 5% CO2 and 85% humidity for 16-18 h. Cells were then treated with various concentrations of GCase-BS molecules for 48 h. Cells were washed once with PBS and lysed by adding distilled water and methanol containing an internal analyte standard. Samples were evaporated to dryness, reconstituted in acetonitrile / water 90 / 10 (v / v) containing 1% DMSO and analyzed by LC-MS / MS. Lipids were simply quantified using the peak area ratio analyte / internal standard (= response).

[0105] To evaluate the cellular localization of the various constructs, immunocytochemical labeling was performed on H4 cells. Cells were seeded in imaging-compatible 96-well plates at 5E3 cells / well and treated with the molecules for 2 h. Afterwards, cells were washed once with PBS and fixed using 4% PFA for 10 min at room temperature. After fixation, cells were washed three times with PBS and blocked with 1% donkey serum, 1 mg / ml saponin, 0.75 mg / ml glycine in PBS for 2 h at room temperature. Primary antibodies were incubated overnight at 4 °C in Ab dilution buffer (0.1% BSA, 1 mg / ml saponin in PBS). After washing three times with PBS, secondary antibodies were incubated in Ab dilution buffer for 2 h at RT. Cells were counterstained with DAPI to label the nuclei and imaged using a 40x objective on an Opera Phenix Plus (Perkin Elmer).

[0106] Evaluation of pharmacokinetics and dynamics of GCase-BS in vivo To determine plasma or brain tissue IgG concentrations in GBA+ / + mice treated with mGCase-mBS, samples were analyzed with a generic ECLIA method specific for human Ig / Fab CH1 / κ domain using a cobas e411 instrument under non-GLP conditions. Prior to analysis, brain tissue samples were mechanically homogenized in 500 μL of tissue extraction buffer containing protease inhibitors using a MagNA Lyser Homogenisator. Briefly, the sample, primary detection antibody (mAb anti-hFab(kappa)), secondary detection antibody (mAb anti-hFab(CH1)) and SA beads were added stepwise to the detection vessel, with each step incubated for 9 min. Finally, the SA bead-bound complex was detected by a measuring cell that counted the number of SA beads in the repeat. The count was proportional to the analyte concentration in the test sample.

[0107] To determine the activity of mGCase-mBS in plasma, samples were analyzed using an artificial substrate. Samples were mixed with 10 mM resorufin-β-glucopyranoside and baseline fluorescence was immediately measured at t0. Accumulation of fluorescent product (=resorufin) was measured after 2 h incubation at 37 °C (λex = 535 nm and λem = 595 nm) and indicated GCase activity. A defined standard curve of active mGCase in 10% matrix was used to determine the amount of active compound in plasma over time.

[0108] Analysis of NfL in mouse plasma Detection of NFL in mouse plasma using Quanterix's digital biomarker detection technology, Simoa®: 38 μl of mouse plasma was mixed with 152 μl of sample diluent from the NF-light Kit (Quanterix #103186) and processed according to the manufacturer's instructions.

[0109] statistical analysis Statistical comparisons of data were performed using GraphPad Prism 6. Parametric tests (two-tailed Student's t-test for pairwise comparisons or ANOVA for multiple comparisons) were used. p- and n-values ​​for all comparisons are shown in the legends of each figure.

[0110] Lyso-IP for proteomic and lipidomic analysis H4 cells (WT and GBA knockout) stably expressing TMEM192-3XHA were seeded in 10 / 15 cm cell culture dishes such that sufficient cells (~20 million for proteomics and ~50 million for lipidomics / replication) were available on the day of lysosome isolation. GBA knockout cells were treated with 1 nM hGCase-hBS for 24 h. On the day of lysosome isolation, cells were washed with ice-cold PBS, gently scraped, and centrifuged at 1000 g for 2 min. The cell pellet was resuspended in 1000 uL of ice-cold PBS and gently lysed using a rotary Dawn homogenizer at medium speed. The homogenate was centrifuged at 1000 g for 2 min to remove cell debris. A portion of the supernatant was saved for quality control analysis. The remaining supernatant (approximately 900ul) was incubated with 500ul of anti-HA magnetic beads (Pierce / Thermo:88836 / 88837) for 20 minutes at room temperature in a rotary shaker. The magnetic beads were separated using a magnetic rack and the flow-through was collected for quality control analysis. The magnetic beads carrying lysosomes were washed with ice-cold PBS. For the proteome samples, 200ul of 1x RIPA buffer was added to the magnetic beads carrying lysosomes and heated at 95°C for 5 minutes. The protein samples obtained from lysosomes were acetone precipitated and used for further analysis. For the lipidome samples, the lysosomes were separated from the magnetic beads using competitive elution due to the presence of high concentration of HA peptide. The magnetic beads carrying lysosomes were incubated with 500ul of 1mg / ml HA peptide (in PBS) and incubated at 37°C for 15 minutes. The magnetic beads were removed using a magnetic rack, and the remaining lysosome-containing sample was immediately frozen at -80°C for further analysis. For the corresponding whole cell lysate samples, cells were seeded in 10 cm culture dishes (approximately 8 million cells per replicate) and treated with hGCase-hBS whenever applicable. Cells were gently scraped and cell pellets were collected by centrifugation at 1000g for 2 min.

[0111] Proteomics Sample Preparation Samples were denatured using Biognosys denaturation buffer, reduced with Biognosys reducing solution for 60 min at 37°C, and alkylated with Biognosys alkylating solution for 30 min at room temperature in the dark. Digestion to peptides was then performed overnight at 37°C using 0.5 μg trypsin (Promega) per sample. Peptides were desalted using C18 MicroSpin plates (The Nest Group) according to the manufacturer's instructions and dried using a SpeedVac system. Peptides were resuspended in 20 μl LC solvent A (1% acetonitrile, 0.1% formic acid (FA)) and Biognosys iRT kit calibration peptides were added. Peptide concentrations were determined using a UV / VIS spectrometer (SPECTROstar Nano, BMG Labtech).

[0112] HRM Mass Spectrometry Acquisition for Proteomics For DIA LC-MS / MS measurements, 1 μg of peptides per sample was injected onto an in-house packed reversed-phase column (75 μm inner diameter, 60 cm length and 10 μm tip PicoFrit emitter from New Objective packed with 1.7 μm charged surface hybrid C18 particles from Waters) on a Thermo Scientific™ EASY-nLC™ 1200 nano liquid chromatography system connected to a Thermo Scientific™ Q Exactive™ HF mass spectrometer equipped with a Nanospray Flex™ ion source. LC solvents were A: 1% acetonitrile in water with 0.1% FA, B: 20% water in acetonitrile with 0.1% FA. The nonlinear LC gradient was 1–59% solvent B for 55 min, followed by 59–90% B for 10 s, 90% B for 8 min, 90%–1% B for 10 s, and 1% B for 5 min, at 60 °C and a flow rate of 250 nl / min. The DIA method consisted of one full-range MS1 scan with 21 DIA segments, adapted from Bruderer et al., 2017.

[0113] Proteomic data analysis Proteins with low intensity and NA values ​​were filtered and subsequently analyzed using the edgeR Bioconductor package. Mapping of protein names to HUGO gene symbols was performed using the Bioconductors org.Hs.eg.db package. Libraries were normalized using TMM to remove compositional bias, and we fitted a negative binomial generalized log-linear model to the log2 intensity of each protein, taking into account genetic, trend and common variance estimates. Tests of differential expression of proteins between comparison groups were tested with a log-likelihood ratio test. For comparison and ranking of interesting hits resulting from contrasts of interest, we introduced a comparison metric that combines significance and changes in protein abundance: metric = -Log10(p-value) × Log2(FC). A metric threshold of 6 was used to filter the top differentially expressed proteins.

[0114] Lipid Extraction for Mass Spectrometric Lipidomics Mass spectrometry-based lipid analysis was performed as described by Lipotype GmbH (Dresden, Germany) (Sampaio et al. 2011). Lipids were extracted using a two-step chloroform / methanol procedure (Ejsing et al. 2009). Samples were analyzed using the following ELISA kits: cardiolipin 14:0 / 14:0 / 14:0 / 14:0 (CL), ceramide 18:1;2 / 17:0 (Cer), diacylglycerol 17:0 / 17:0 (DAG), hexosylceramide 18:1;2 / 12:0 (HexCer), lysophosphatidic acid 17:0 (LPA), lysophosphatidylcholine 12:0 (LPC), lysophosphatidylethanolamine 17:1 (LPE), lysophosphatidylglycerol 17:1 (LPG), lysophosphatidylinositol 17:1 (LPI), lysophosphatidylserine 17:1 (LPS), phosphatidate 17:1 (LPG), lysophosphatidylinositol 17:1 (LPI), lysophosphatidylserine 17:1 (LPS), phosphatidic acid 17:0 (LPA), lysophosphatidylcholine 12:0 (LPC), lysophosphatidylethanolamine 17:1 (LPE), lysophosphatidylglycerol 17:1 (LPG), lysophosphatidylinositol 17:1 (LPI), lysophosphatidylserine 17:1 (LPS), phosphatidic acid ... The mixture was spiked with an internal lipid standard mixture containing phosphatidylcholine 17:0 / 17:0 (PA), phosphatidylethanolamine 17:0 / 17:0 (PE), phosphatidylglycerol 17:0 / 17:0 (PG), phosphatidylinositol 16:0 / 16:0 (PI), phosphatidylserine 17:0 / 17:0 (PS), cholesterol ester 20:0 (CE), sphingomyelin 18:1;2 / 12:0;0 (SM), triacylglycerol 17:0 / 17:0 / 17:0 (TAG) and cholesterol D6 (Chol). After extraction, the organic phase was transferred to an injection plate and dried in a speed vacuum concentrator. The dried extract from the first step was resuspended in 7.5 mM ammonium acetate in chloroform / methanol / propanol (1:2:4, V:V:V), and the dried extract from the second step was resuspended in a 33% ethanolic solution of methylamine in chloroform / methanol (0.003:5:1; V:V:V). All liquid handling steps were performed using a Hamilton Robotics STARlet robot platform equipped with anti-droplet control for organic solvent pipetting. MS Data Acquisition for Lipidomics

[0115] Samples were analyzed by direct infusion on a QExactive mass spectrometer (Thermo Scientific) equipped with a TriVersa NanoMate ion source (Advion Biosciences). Samples were analyzed in both positive and negative ion modes in a single acquisition with a resolution of Rm / z = 200 = 280000 for MS experiments and Rm / z = 200 = 17500 for MSMS experiments. MSMS was triggered by an inclusion list encompassing the corresponding MS mass range scanned in 1 Da increments (Surma et al. 2015). Both MS and MSMS data were combined to monitor CE, DAG and TAG ions as ammonium adducts, PC, PC O- as acetate adducts, and CL, PA, PE, PE O-, PG, PI and PS as deprotonated anions. Using MS alone, we monitored LPA, LPE, LPE O−, LPI and LPS as deprotonated anions, Cer, HexCer, SM, LPC and LPC O− as acetate adducts, and cholesterol as the ammonium adduct of the acetylated derivatives (Liebisch et al. 2006).

[0116] Lipidomic Data Analysis Data were analyzed using an in-house developed lipid identification software based on LipidXplorer (Herzog et al. 2012, 2011). Post-processing and normalization of data were performed using an in-house developed data management system. For further data analysis, only lipid identifications with a signal-to-noise ratio >5 and a signal intensity 5-fold higher than the corresponding blank sample were considered. Data were analyzed in R version 4.0.3 (2020-10-10) (RCore Team 2017) using the tidyverse package (version 1.3.0) (Wickham 2019) and bioconductor pcaMethods (Stacklies et al. 2007). Lipids were quantified in molar fractions (molp) and normalized to the total lipid amount per sample due to the large differences in the total lipid amount between samples. An occupancy threshold of 70% was applied, resulting in 1196 lipids to be compared. Differential lipidomics analysis was performed using unpaired t-tests between test groups (GBA-KO vs. WT and KOE vs. KO in both lysosomal and whole cell lysates). Fold changes between comparison groups are defined as the average Log2 fold change. We used the same combined p-value / logFC metric as for lipid contrasts to compare the top hits. To display the top hits between GBA-KO vs. WT and KOE vs. KO comparisons (Figure 4D, Supplementary Figure B), we applied a metric threshold of 1 in both directions.

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Claims

1. A fusion protein comprising: a) a lysosomal protein; b) an Fc region of an antibody; and and c) an antibody fragment that targets the transferrin receptor, said antibody fragment having a monovalent binding mode.

2. The fusion protein according to claim 1, wherein the lysosomal protein is a β-glucocerebrosidase (Gcase) protein, preferably a human Gcase protein or a variant thereof.

3. The fusion protein according to claim 1 or 2, wherein the Fc region of an antibody is the Fc region of an IgG antibody, preferably an IgG1 antibody.

4. The fusion protein of claims 1 to 3, wherein the Fc region lacks Fc receptor gamma binding.

5. The antibody fragment that targets the transferrin receptor is selected from the group consisting of Fv, Fab, Fab', Fab'-SH, F(ab') 、 The fusion protein according to claims 1 to 4, which is selected from the group consisting of diabodies, linear antibodies, single chain antibody molecules such as scFv, scFab, cross-Fab and single domain antibodies (dAb).

6. The fusion protein of claims 1 to 5, wherein one chain of the Fc region is fused at its N-terminus to the C-terminus of the lysosomal protein and a second Fc chain is fused at its C-terminus to the antibody fragment that targets the transferrin receptor.

7. The fusion protein according to claims 1 to 6, wherein the two Fc chains form a dimer using knob-into-hole technology.

8. The fusion protein comprises two protein chains:

3. A first protein chain comprising the lysosomal protein fused at its C-terminus to a first chain of the Fc region comprising the knob-into-hole technology; 4. A second protein chain comprising a second chain of the Fc region comprising the knob-into-hole technology fused at its C-terminus to the scFab antibody fragment targeting the transferrin receptor; The fusion protein according to claims 1 to 7, comprising:

9. The fusion protein according to claims 1 to 8, wherein the human Gcase protein has the amino acid sequence shown in SEQ ID NO:

1.

10. 9. The fusion protein of claim 8, wherein the first protein chain has the amino acid sequence set forth in SEQ ID NO:2 and the second single chain protein has the amino acid sequence set forth in SEQ ID NO:

3.

11. An isolated nucleic acid molecule encoding a fusion protein according to claims 1 to 11.

12. The isolated nucleic acid of claim 11 , wherein the nucleic acid is a circular RNA.

13. A host cell comprising the isolated nucleic acid molecule of claim 11 or 12.

14. A pharmaceutical formulation comprising the fusion protein according to claims 1 to 10.

15. A fusion protein according to claims 1 to 10 for use as a medicament.

16. A fusion protein according to claims 1 to 10 for use in the treatment of a neurodegenerative disorder.

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