Background of the Invention

The invention relates to conjugates including an a-L-iduronidase enzyme and a dendrimeric targeting moiety and the use of such conjugates in the treatment of disorders that result from a deficiency of that enzyme, such as mucopolysaccharidosis type I (MPS-I).

Lysosomal storage disorders are group of about 50 rare genetic disorders in which a subject has a defect in a lysosomal enzyme that is required for proper metabolism. These diseases typically result from autosomal or X-linked recessive genes. As a group, the incidence of these disorders is about 1 :5000 to 1 : 10,000.

MPS-I is a lysosomal storage disorder that results from a deficiency of a-L-iduronidase (IDUA), an enzyme that is required for lysosomal degradation of glycosaminoglycans (GAGs). IDUA removes sulfate from sulfated a-L-iduronic acid, which is present in two GAGs, heparan sulfate and dermatan sulfate. Those with the disorder are unable to break down and recycle these GAGs. This deficiency results in the buildup of GAGs throughout the body, which has serious effects on the nervous system, joints, and various organ systems including heart, liver, lung, and skin. There are also a number of physical symptoms, including coarse facial features, enlarged head and abdomen, and skin lesions. In the most severe cases, the disease can be fatal before age 10 and is accompanied by severe mental retardation.

MPS-I is divided into three subtypes based on severity of symptoms, MPS-I H, also called Hurler syndrome or α-L-iduronidase deficiency, is the most severe form; MPS-I S, also called Sheie syndrome, is the mildest form; and MPS-I H-S, also called Hurler-Sheie syndrome, is less severe than Hurler syndrome alone.

The most severe disease can result from a complete loss of IDUA activity. Severe disease is characterized by mental decline, reduction in height, enlarged organs, facial features such as flat face, depressed nasal bridge, and bulging forehead, and organ and bone enlargement. Death often results before age 10 due to respiratory problems, such as obstruction or infection, or cardiac complications.

In moderate cases, symptoms become apparent between ages 3 and 8. These individuals may have moderate mental retardation and learning difficulties, short stature, marked smallness in the jaws, progressive joint stiffness, compressed spinal cord, clouded corneas, hearing loss, heart disease, coarse facial features, and umbilical hernia. Respiratory problems, sleep apnea, and heart disease may develop in adolescence. Life expectancy is generally into the late teens or early twenties.

In mild cases, cognitive decline is not detected or mild, and symptoms begin to appear after age 5. Some of the peripheral symptoms, such as glaucoma, retinal degeneration, clouded corneas, carpal tunnel syndrome or other nerve compression, stiff joints, claw hands and deformed feet, a short neck, and aortic valve disease, obstructive airway disease, and sleep apnea.

Over 100 different mutations causing MPS-I have been identified. Most of these mutations are missense or nonsense mutations. VV402X and Q70X are the most common in Caucasian populations.

The α-L-iduronidase activity levels in families in which the Hurler syndrome is present has been studied. Samples of affected patients, heterozygotes (asymptomatic carriers), and normal subjects were found to be clearly distinguished by α-L-iduronidase activity alone. For example, in mixed leukocyte preparations studied by Wappner et al. , Pediat. Res. 10:629-632, 1976, Hurler patients had 0 to 3% activity, heterozygotes 19 to 60% activity, and normal subjects 83 to 121 % of the mean normal activity. These studies indicate that correction of enzymatic activity and/or GAG levels to the level found in asymptomatic heterozygotes is the threshold that would likely result in a clinically effective therapy.

There is no cure for MPS-I. In addition to palliative measures, therapeutic approaches have included bone marrow grafts and enzyme replacement therapy. While bone marrow grafts have been observed to improve outcomes in MPS-I patients, patients undergoing this procedure are at substantial risk of development of graft rejection (e.g. , graft-versus-host disease) or even death. Enzyme replacement therapy by intravenous administration of IDUA has also been shown to have benefits, including improvement in organs such as liver, heart, and lung, as well as various physical tests. Like bone marrow grafts, this approach is not expected to have significant effects on central nervous system deficits associated with MPS-I because the enzyme does not cross the blood-brain barrier (BBB).

Methods for increasing delivery of IDUA to the brain have been and are being investigated, including intrathecal delivery. Intrathecal delivery, however, is a highly invasive technique.

Less invasive and more effective methods of treating MPS-I that address the neurological disease symptoms, in addition to the other symptoms, would therefore be highly desirable.

Summary of the Invention

The present invention is direct to compounds that include a dendrimeric targeting moiety (e.g. , a targeting moiety including a LRP1 targeting ligand such as Angiopep-2) and an enzyme having a-L- iduronidase activity. These compounds are exemplified by IDUA-Angiopep-2 conjugates which can be used to treat MPS-I. Because these conjugates are capable of crossing the blood-brain barrier (BBB), they can treat not only the peripheral disease symptoms, but can also be effective in treating CNS symptoms. In addition, because targeting moieties such as Angiopep-2 are capable of targeting enzymes to the lysosomes, these conjugates are predicted to be more effective than the enzyme alone in treating peripheral disease symptoms, as well as CNS symptoms.

Accordingly, in a first aspect, the invention features a compound having the structure:

A-(L -B) _n

Formula I

wherein A is an enzyme having a-L-iduronidase activity;

L is absent or a linker; and

B is a dendrimeric targeting moiety;

n is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, or 13.

In some embodiments, the dendrimeric targeting moiety has the structure:

wherein m is 1 , 2, 3, 4, 5, or 6;

L ² is absent or a linker; and

C includes a LRP1 ligand.

In some embodiments, L ² is absent.

In other embodiments, C includes a polypeptide having an amino acid sequence at least 70% (e.g. , at least 75%, 80%, 85%, 90%, 95%, 99%, or 100%) identical to Angiopep-1 (SEQ ID NO: 1 TFFYGGCRGKRNNFKTEEY), Angiopep-2 (SEQ ID NO:2 TFFYGGSRGKRNNFKTEEY), Angiopep-2-4D (SEQ ID NO:3 TFFYGGSrGkrNNFkTEEY, wherein the lowercase symbols represent D-amino acids), Angiopep-7 (SEQ ID NO:4 TFFYGGCRGRRNNFRTEEY), reverse Angiopep-2 (SEQ ID NO:5

YEETKFNNRKGRCGGYFFT), or reverse Angipep-2 all D (SEQ ID NO:6 yeetkfnnrkgrcggyfft, wherein the lowercase symbols represent D-amino acids).

In certain embodiments, C includes Angiopep-1 , Angiopep-2, or Angiopep-2-4D.

In some embodiments, C includes Angiopep-2.

In certain embodiments, C has the structure:

In some embodiments, m is 3.

In other embodiments, L is click-chemistry linker such as a monofluorocyclooctyne (MFCO) linker, a difluorocyclooctyne (DFCO) linker, a dibenzocyclooctyne (DBCO) linker, a cyclooctyne (OCT) linker, a biarylazacyclooctyne (BARAC) linker, a difluorobenzocyclooctyne (DIFBO) linker, or a bicycle[6.1.0]nonyne (BCN) linker. In certain embodiments, linker having the structure:

In some embodiments, the enzyme is attached via one or more NH groups derived from reaction of a primary amine group of the enzyme (e.g. , the ε-amine of a lysine residue of the enzyme).

In certain embodiments, the compound has the structure:

Formula III

In some embodiments, n is 1 or 2.

In other embodiments, transcytosis and/or endocytosis of the compound is predominantly mediated by LRP1.

In certain embodiments, the compound has increased (e.g. , an increase by 100%, 150%, 200%, 300%, 400%, 500%, 600%. 700%, 800%, 900%, 1000% or more, or an increase by more than 1.2-fold, 1.4-fold, 1.5-fold, 1.8-fold, 2.0-fold, 3.0-fold, 3.5-fold, 4.5-fold, 5.0-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 1000-fold, or more) enzymatic activity in vitro and/or in vivo compared to the unconjugated enzyme.

In some embodiments, the compound has increased (e.g. , an increase by 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more, or an increase by more than 1.2-fold, 1.4-fold, 1.5-fold, 1.8-fold, 2.0-fold, 3.0-fold, 3.5-fold, 4.5-fold, 5.0-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 1000-fold, or more) enzymatic activity in the brain compared to the unconjugated enzyme.

In other embodiments, the compound has increased (e.g. , an increase by 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 100% or more, or an increase by more than 1.2-fold, 1.4-fold, 1.5-fold, 1.8-fold, 2.0-fold, 3.0-fold, 3.5-fold, 4.5-fold, 5.0-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 1000-fold, or more) stability in vitro and/or in vivo compared to the unconjugated enzyme.

In certain embodiments, the compound is capable of reducing (e.g., a decrease by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more; or a decrease by 0.01- fold, 0.02-fold, 0.1 -fold, 0.3-fold, 0.5-fold, 0.8-fold, 0.9-fold, 0.95-fold or more) glycosaminoglycan (GAG) levels in vitro and/or in vivo in a model of MPS-I.

In some embodiments, the compound is capable of reducing (e.g. , a decrease by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more; or a decrease by 0.01- fold, 0.02-fold, 0.1 -fold, 0.3-fold, 0.5-fold, 0.8-fold, 0.95-fold or more) GAG levels in vitro and/or in vivo in a model of MPS-I to the level of a model of a heterozygote carrier of MPS-I.

In other embodiments, the compound is capable of reducing (e.g. , a decrease by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more; or a decrease by 0.01- fold, 0.02-fold, 0.1 -fold, 0.3-fold, 0.5-fold, 0.8-fold, 0.9-fold , 0.95-fold or more) GAG levels in vitro and/or in vivo in a model of MPS-I to the level of a model of a-L-iduronidase activity in a healthy subject.

In another aspect, the invention features a pharmaceutical composition including any of the foregoing compounds and a pharmaceutically acceptable excipient.

In another aspect, the invention features a method of treating or treating prophylactically a subject having MPS-I. This method includes administering to the subject an effective amount of any of the foregoing compounds or pharmaceutical compositions.

In some embodiments, an effective amount is an amount capable or reducing GAG levels in the subject to the level of a heterozygote carrier of MPS-I. In other embodiments, an effective amount is an amount capable of increasing enzymatic activity in the subject to the level of a heterozygote carrier of MPS-I.

In certain embodiments, an effective amount is an amount capable of reducing GAG levels in the subject to the levels of a normal subject (e.g. a subject with normal IDUA enzymatic activity). In other embodiments, an effective amount is an amount capable of increasing enzymatic activity in the subject to the level of a normal subject.

In some embodiments, the subject has neurological symptoms.

In other embodiments, the subject starts treatment under five years of age (e.g. , under four years of age, under three years of age, under two years of age, under one year of age). In certain

embodiments, the subject is an infant (e.g. , the subject is between 1 month and 12 months of age).

In some embodiments, administering includes parenteral administration (e.g. , intravenous administration). In other embodiments, administering does not include intrathecal or intracranial administration.

In chemical structures the symbol " ·~>~ " represents the point of attachment of the structure to the rest of the conjugate. In some embodiments, the chemical structure is attached to the rest of the conjugate via a single bond at the point of attachment.

As used herein, "an enzyme having a-L-iduronidase activity" refers to any protein (e.g. , IDUA, a fragment, or analog thereof) that is capable of breaking down unsulfated a-L-iduronic acid and is useful in the treatment of MPS-I.

As used herein, "click-chemistry linker" refers to a linking group that is formed by the reaction between a click-chemistry reaction pair. By "click-chemistry reaction pair" is meant a pair of reactive groups that participates in a modular reaction with high yield and a high thermodynamic gain, thus producing a click-chemistry linker. As used herein, "dendrimeric targeting moiety" refers to a branched macromolecule including a LRP1 ligand having a core moiety with at least three functional groups.

As used herein, "LRP1 ligand" refers to any compound (e.g. , a polypeptide) capable of specifically binding LRP1. LRP1 or low density lipoprotein receptor-related protein 1 , also known as alpha-2-macroglobulin receptor (A2MR), apolipoprotein E receptor (APOER), or cluster of differentiation (CD91 ) is a protein forming a receptor found in the plasma membrane of cells involved in receptor- mediated endocytosis. In humans, the LRP1 protein is encoded by the LRP1 gene. By "specifically binds" is meant binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a binding agent is competitively inhibited by excess unlabeled target. The term "specific binding," "specifically binding," or "specifically binds to" a particular protein as used herein can be exhibited, for example, by a molecule having a K _D for the target of 10 ^"4 M or lower, alternatively 10 ^"5 M or lower, alternatively 10 ^"6 M or lower, alternatively 10 ^"7 M or lower, alternatively 10 ^"8 M or lower, alternatively 10 ^"9 M or lower, alternatively 10 ^"10 M or lower, alternatively 10 ^"11 M or lower, alternatively 10 ^"12 M or lower, or a K _D in the range of 10 ^"4 M to 10 ^"12 M or 10 ^"6 M to 10 ^"10 M or 10 ^"7 M to 10 ^"9 M. As will be appreciated by the skilled artisan, affinity and K _D values are inversely related. A high affinity for a target is measured by a low K _D value.

As used herein "transcytosis and/or endocytosis of a compound is predominantly mediated by

LRP1 " means transcytosis and/or endocytosis is inhibited by the LRP1 inhibitor, receptor associated protein (RAP), by a greater percentage inhibition than by mannose-6-phosphate ( M6P), each as compared to transcytosis and/or endocytosis in the absence of both RAP and M6P, respectively. Brief Description of the Drawings

Figures 1A-1 C are graphs illustrating the in vitro enzymatic activity of unconjugated IDUA,

IDUAL7, and IDUAL17 over 2 to 96 hours post incubation.

Figure 1 D is an image illustrating the in vitro enzymatic activity of unconjugated IDUA, IDUAL7, and IDUAL17.

Figure 2 is a graph illustrating the reduction of glycosaminoglycan (GAG) levels to normal levels by IDUAL17 in MPS-I fibroblasts.

Figure 3 is a graph illustrating the effect of M6P and RAP on the in vitro uptake of unconjugated IDUA, IDUAL7, and IDUAL17.

Figures 4A-4E are images illustrating binding of unconjugated IDUA, IDUAL7, and IDUAL17 to LRP1.

Figures 5A and 5B are graphs illustrating the in vivo enzymatic activity of IDUAL17 in the brain and liver.

Figures 6A and 6B are graphs illustrating the in vivo reduction of GAG levels in the brain and liver by IDUAL17.

Detailed Description

The present invention is related to conjugates that include an enzyme having a-L-iduronidase activity and a dendrimeric targeting moiety (e.g. , a targeting moiety including Angiopep-2) joined by a linker (e.g. , a click-chemistry linker). The targeting moiety is capable of transporting the enzyme to the lysosome and/or across the blood-brain barrier (BBB). Such compounds are exemplified by Angiopep-2- IDUA conjugates. These conjugates maintain IDUA enzymatic activity in an enzymatic assay, in a cellular model of MPS-I, and in an in vivo model of MPS-I. Because targeting moieties including Angiopep-2 are capable of transporting proteins across the BBB, these conjugates are expected to have not only peripheral activity, but also have activity in the central nervous system (CNS). In addition, targeting moieties including Angiopep-2 are taken up by cells that express the LRP1 receptor into lysosomes. Accordingly, we predict that these targeting moieties can increase enzyme concentrations in the lysosome, thus resulting in more effective therapy, particularly in tissues and organs that express the LRP1 receptor, such as liver, kidney, and spleen.

These features overcome some of the biggest disadvantages of current therapeutic approaches because intravenous administration of IDUA alone does not result in effective CNS delivery. In contrast to physical methods for bypassing the BBB, such as intrathecal or intracranial administration, which are highly invasive and thus generally an unattractive solution to the problem of CNS delivery, the present invention allows for effective noninvasive delivery of IDUA to the brain. In addition, improved transport of the therapeutic to the lysosomes may allow for reduced dosing and/or reduced frequency of dosing, as compared to standard enzyme replacement therapy. a-L-iduronidase

The conjugates of the present invention include an enzyme having a-L-iduronidase activity, e.g. ,

IDUA, or an analog or fragment thereof having α-L-iduronidase activity, that is useful for treating MPS-I. The compounds may include IDUA, a fragment of IDUA that retains enzymatic activity, or an IDUA analog. IDUA sequences may include an amino acid sequence substantially identical (e.g. , at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical) to the sequence of mature human IDUA and which retains enzymatic activity.

The sequence of human IDUA is shown below:

SEQ ID NO: 7 - MRPLRPRAAL LALLASLLAA PPVAPAEAPH LVHVDAARAL WPLRRFWRST

GFCPPLPHSQ ADQYVLSWDQ QLNLAYVGAV PHRGIKQVRT HWLLELVTTR GSTGRGLSYN FTHLDGYLDL

LRENQLLPGF ELMGSASGHF TDFEDKQQVF EWKDLVSSLA RRYIGRYGLA HVSKWNFETW NEPDHHDFDN

VSMTMQGFLN YYDACSEGLR AAS PALRLGG PGDSFHTPPR SPLSWGLLRH CHDGTNFFTG EAGVRLDYIS

LHRKGARSS I SILEQEKWA QQIRQLFPKF ADTPIYNDEA DPLVGWSLPQ PWRADVTYAA MWKVIAQHQ

NLLLANTTSA FPYALLSNDN AFLSYHPHPF AQRTLTARFQ VNNTRPPHVQ LLRKPVLTAM GLLALLDEEQ

LWAEVSQAGT VLDSNHTVGV LASAHRPQGP ADAWRAAVLI YASDDTRAHP NRSVAVTLRL RGVPPGPGLV

YVTRYLDNGL CSPDGEWRRL GRPVFPTAEQ FRRMRAAEDP VAAAPRPLPA GGRLTLRPAL RLPSLLLVHV

CARPEKPPGQ VTRLRALPLT QGQLVLVWSD EHVGSKCLWT YEIQFSQDGK AYTPVSRKPS TFNLFVFS PD

TGAVSGSYRV RALDYWARPG PFSDPVPYLE VPVPRGPPSP GNP.

Mature IDUA is formed by the cleavage of the N-terminal 26 amino acids from the full length sequence.

Fragments of IDUA that display enzymatic activity are known in the art. To test whether particular fragment or analog has enzymatic activity, the skilled artisan can use any appropriate assay. Assays for measuring IDUA activity, for example, are known in art, including those described in Hopwood et al. , Clin. Sci. 62: 193-201 , 1982 and Hopwood et al. , Clin. Chim. Acta 92:257-65, 1979. One assay is described below. Using any of these assays, the skilled artisan can determine whether a particular IDUA fragment or analog has enzymatic activity.

In certain embodiments, an IDUA fragment is used. IDUA fragments may be at least 50, 100,

150, 200, 250, 300, 350, 400, 450, or 500 amino in length. In certain embodiments, the enzyme may be modified, e.g. , using any of the polypeptide modifications described herein such as replacing one or more amino acids with a cysteine to add a site for conjugation.

Significant work has been performed to elucidate structure-function relationships between IDUA mutations and function of the IDUA enzyme. To this end, the catalytic region of IDUA has been predicted based on conservation between related proteins, as described in Henrissa et al. , Proc. Natl. Acad. Sci. USA 92:7090-4, 1995. In addition, a homology model, based on the crystal structure of structure of a related protein β-xylosidase from Thermoanerobacterium saccharolyticum has been created and has led to an understanding of why certain mutants produce either minor or severe changes to protein structure and thus contribute to whether the individual having that mutation exhibits attenuated or severe disease.

Other studies have shown that mutations associated with severe cases tend to affect a greater number of atoms in IDUA than those associated with attenuated cases. Recent work has also suggested that that the C-terminal of IDUA may be important for clinical manifestations, as described in Vanza et al., Am. J.

Med. Genet. A 149A:965-74, 2009. This work therefore provides a relationship between the structure of

IDUA and its function and allows for the development of IDUA fragments and ana logs which retain enzymatic activity for use in the conjugates and methods of the invention.

Targeting Moieties

Dendrimers

A dendrimer is a branched macromolecule having a core moiety with at least three functional groups. A dendrimer can have multiple branch moieties that are attached to the core moiety, and the surface branch moieties can be functionalized for attachments of various molecules (e.g. , LRP1 ligands such as Angiopep-2). One advantage of using a dendrimer is the availability of multiple surface functionalities to which multiple molecules e.g. , LRP1 ligands such as Angiopep-2) can be conjugated.

Dendrimer

The core moiety of a dendrimer can be any known in the art, including those selected from the group consisting of propargylamine, ethylenediamine, triethanolamine, pentaerythritol, azido- propyl(alkyl)amine, hydroxyethyl(alkyl)amine, tetraphenyl methane, trimesoylchloiride, diamino hexane, diaminobutane, cystamine, and propylenediamine, in case of PAMAM dendrimers. The core moiety can also be propyleneamine in which case the dendrimer is poly(propyleneamine) (POPAM). Alternatively, the core moiety can be lysine, in which case the dendrimer is poly-lysine. Typically core moieties can have 1 to 12 branches (e.g. , 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , or 12 branches). The core moieties are functionalized to form reactive groups (e.g. , by reacting to methyl acrylate) for addition of the branch moieties. One or branch moieties can be attached to the core moiety via the functional groups. LRP1 ligands and enzymes can also be attached to the core moiety with or without linkers via the functional groups.

The branch moieties form successive layers around the core moiety, and are also referred to as

"generations" in the art. Each branch moiety attached to a branch of the core moiety can have 2 to 8 branches (2, 3, 4, 5, 6, 7, or 8 branches). The branch moieties can be the same as the core moieties, can be a derivative of the core moiety, or can be selected from the group consisting of propargylamine, ethylenediamine, triethanolamine, pentaerythritol, azido-propyl(alkyl)amine, hydroxyethyl(alkyl)amine, tetraphenyl methane, trimesoylchloride, diamino hexane, diaminobutane, cystamine, propylenediamine, proplyleneamine, and lysine.

A dendrimer can have 2 to 10 layers (2, 3, 4, 5, 6, 7, 8, 9, or 10 layers) of branches, terminating in the outer most branch moieties, which are also referred to as surface branches. The surface branches can be functionalized for attachment of multiple chemical entities (e.g. , LRP1 ligands such as Angiopep- 2). The number of surface branches is computed by the formula, n = p(b , where n = the number of surface branches, b= the number of branches each branch moiety has, and / = the number of successive layers of branches of the dendrimer. Since a dendrimer will be attached to multiple LRP1 ligands and one or more enzymes, it is desirable that the dendrimer size be in a range to accommodate attachment of these cargoes. For example, a desirable dendrimer molecular weight is less than 500 kilodaltons (e.g. , 10, 50, 100, 200, 300, or 500 kilodaltons).

PAMAM is perhaps the most well known dendrimer. The core of PAMAM is a diamine

(commonly ethylenediamine), which is reacted with methyl acrylate, and then another ethylenediamine to make the generation-0 (G-0) PAMAM. Successive reactions create higher generations, which tend to have different properties. Lower generations are generally flexible molecules with no appreciable inner regions, while medium sized (G-3 or G-4) have internal space that is essentially separated from the outer shell of the dendrimer. Very large (G-7 and greater) dendrimers are generally more like solid particles with very dense surfaces due to the structure of their outer shell.

Synthesis of dendrimers

Methods for synthesizing dendrimers are well known in the art, as described herein, and the branched portion of the dendrimer (the X _∞re and X portions) can also be purchased from a commercial supplier with varying numbers of layers of branches. There are two commonly used methods of dendrimer synthesis: divergent synthesis and convergent synthesis. In divergent synthesis (shown below), the dendrimer is assembled from a multifunctional core, which is extended outward by a series of reactions, commonly a Michael reaction. Each step of the reaction is generally driven to full completion to prevent mistakes in the dendrimer, which can cause trailing generations (some branches are shorter than the others). Such impurities can impact the functionality and symmetry of the dendrimer, but are extremely difficult to purify out because the relative size difference between perfect and imperfect dendrimers is very small. γΥ _γ γΥΥγ _γ

^{' γ} Υγ _Ν γ

γ Υ

ΐ Υ Υ

Initiator γ γ ^γ

core Υ γ γ ^γ γ ' i '

ΥΥγγγΥ

In convergent synthesis (shown below), dendrimers are built from small molecules that end up at the surface of the sphere, and reactions proceed inward, such that the inward most molecules that are attached last are attached to a core. This method makes it much easier to remove impurities and shorter branches along the way, so that the final dendrimer is more monodisperse. However dendrimers made this way are not as large as those made by divergent methods because crowding due to steric effects along the core is limiting.

l point

Alternatively, dendrimers can also be synthesized by click-chemistry, employing Diels-Alder reactions, thiol-yne reactions, and azide-alkyne reactions.

A dendrimer can be synthesized to have different functionalities in the core and the branches to control properties such as solubility, thermal stability, and attachment of compounds for particular applications. Synthetic processes can also precisely control the size, number of branches, numbers of layers of branches from the core, and the functionalities of the terminal branches for attachment of various reactive groups.

Conjugation of LRP1 ligands to surface branches of dendrimers

The surface branches of dendrimers can be functionalized for conjugation of LRP1 ligands derivatized with appropriate reactive groups. For example, the surface branches can be reacted with compounds, e.g. , N -succinimidyl 3-(2-pyridy Idithio) (SPDP) to generate a dendrirner-pyridyl-disulfide intermediate that can be then be reacted with polypeptides containing a cysteine residue. Alternatively, the surface branches of dendrimers can be reacted with N-succinimidyl S-acetylthioacetate (SATA) to form a dendrimer-sulfydryl intermediate that can be reacted with a maleimide derivatized polypeptide. SATA is reactive towards amines and adds protected sulfhydryls groups), and BMOE (bis- maleimidoethane). Linkers can be used to conjugate polypeptides to the surface functionalities of dendrimers and are described below.

Dendrimer Configurations Each part of a given conjugate, including the enzyme, linker, and LRP1 ligand, can be selected independently. That is, insofar as the interacting chemical substituents are compatible with one another, any of the linkers described herein can be used to conjugate any of the LRP1 ligands and enzymes described. The conjugates can then be used to deliver the enzymes to a patient for treatment of MPS-I. With the inclusion of a detectable marker, the present conjugates can also be used as imaging agents, providing the means to map the distribution of the enzymes and/or the receptors for which the conjugates have affinity.

While specific configurations are discussed further below, we note that a given conjugate can include one or more LRP1 ligands relative to each enzyme (e.g. , 1-4 LRP1 ligands relative to each enzyme within the conjugate) and one or more enzymes relative to the LRP1 ligands (e.g. , 1 -4 enzymes per LRP1 ligand). As noted, a given conjugate is likely to include a single enzyme, but it may include two or more (e.g. , 2, 3, or 4) that are identical to one another or different from one another. The component parts of the present conjugates can be configured in a variety of ways. Overall, the conjugate can assume an essentially linear form with an enzyme being linked to at least one LRP1 ligand, which is in turn linked to at least one enzyme. Alternatively, the conjugate can have a branched configuration as seen in dendrimers, with one or more branches extending at some point from enzyme (D). Where the present conjugates include a branched portion, we may refer to the conjugate as a "dendrimer conjugate" or "dendrimeric conjugate" with the understanding that the inclusion of the enzyme does not allow for a fully symmetrical dendrimeric form. A dendrimeric conjugate can be structured as in Formula IV:

/

Formula IV

D is an enzyme that is linked to the core moiety of the dendrimer conjugate (X _∞re ) either directly (e.g., by way of a bond between the enzyme and X _∞re ) or indirectly (e.g., by way of a bifunctional linker that joins the enzyme to X _∞re ). As X _∞re and _bmn ch both join one part of a conjugate to another, we may also refer to either moiety more simply as a "linker". The complexity of the core moiety can vary, with the number of available extension points, p, varying from 2 to 6, inclusive. Each extension point p can terminate in (and be joined to) a branch moiety, X _bra nch, that, like X _∞re , varies in complexity with each branch having from 2 to 4 branches, b. X _n ^th is one of n surface branches, and /, an integer from 1 to 5, inclusive, is the number of successive layers of _bmn ch moieties. Where / is 1 , each _bmn ch is attached to X _∞ re- Where / is more than 1 , each _bmn ch distal to the first _bmn ch is attached to another X _bmn ch- With regard to the surface branches, X _n ^th is one of n surface branches of the dendrimer. n = p(b'), and n is typically <512 (e.g. , <500, <400, <300, <200, <50, <10, or <8 branches). To illustrate: where there are two extension points p, where / is 1 , and where there are two branches b from each X _bra nch, X _n ' ^h is 4; where there are three extension points p, where / is 1 , and where there are three branches b from each Xbranc , X _n ^th is 9; and so forth. A _m is a LRP1 ligand as described herein that is attached to a surface branch X _n ^th . The number of LRP1 ligands A _m is less than or equal to the number of surface branches, as each surface branch can be joined to a LRP1 ligand, and some surface branches can be either free of any additional components or joined directly to an enzyme D' {i.e. , at some surface branches, the LRP1 ligand represented by A _m is absent). The enzyme D' is attached to one or more A _m or, as noted, may replace one or more (but not all) A _m , attaching directly to one or more X _n ^th . The number of D' in the dendrimer conjugate can be up to three times the number of LRP1 ligands, as up to three enzymes can be joined to each LRP1 ligand. The molecular weight of the dendrimer, excluding D, D' and A _m , is < 500 kilodalton (e.g. , < 500, < 400, < 300, < 200, < 100, < 50, or <20 kilodaltons).

The linkers employed as X _∞re and X _b mnch can be the same or different, and one can make less complex dendrimer conjugates by employing a bifunctional linker as either X _∞re or Xbranch- Where X _∞re is a bifunctional linker, p is 1 and the complexity that would have been generated by multiple extensions from X _∞re is missing. This arrangement is illustrated in Formula V below, with the remainder of the conjugate as described above.

Formula V

In a variant of this configuration, X _∞re is absent, in which case the enzyme is joined directly to an branch- Where branch . rather than X _∞re , is a bifunctional linker, b is 1 , and the complexity that would have been generated by multiple extensions from _bra nch is missing. This arrangement is illustrated in Formula VI below, with the remainder of the conjugate as described above.

/

Formula VI

One advantage of the dendrimeric conjugate is the inclusion of multiple surface functionalities to which multiple LRP1 ligands and/or enzymes can be conjugated. The ability to alter the complexity of the dendrimeric conjugate allows one to accommodate the various component parts of the conjugate. Where X _∞re and Xbranch are both bifunctional linkers, the conjugate is linear, not dendrimeric.

The dendrimer part of the compound may include a core moiety selected from the group consisting of propargylamine, ethylenediamine, triethanolamine, pentaerythritol, azido-propyl(alkyl)amine, hydroxyethyl(alkyl)amine, tetraphenyl methane , trimesoylchloride, diamino hexame, diaminobutane, cystamine, and propylenediamine. These cores are typically used to synthesize the poly(amido amine) (PAMAM) dendrimer. Lysine can also be used as a core moiety to synthesize a polylysine dendrimer. Alternatively the compound can include a propyleneimine to synthesize a POPAM dendrimer.

The conjugate of the invention can have branch moieties selected from the group consisting of propargylamine, ethylenediamine, triethanolamine, pentaerythritol, propylamine, propyleneimine, azido- propyl(alkyl)amine, hydroxyethyl(alkyl)amine, tetraphenyl methane, trimesoylchloride, diamino hexane, diaminobutane, cystamine, propylenediamine, and lysine. Alternatively, the branch moieties can be derivatives of any one of propargylamine, ethylenediamine, triethanolamine, pentaerythritol, propylamine, propyleneimine, azido-propyl(alkyl)amine, hydroxyethyl(alkyl)amine, tetraphenyl methane,

trimesoylchloride, diamino hexane, diaminobutane, cystamine, propylenediamine, and lysine.

One or more terminal branches on the surface of the dendrimer can be functionalized to attach various numbers of LRP1 ligands (e.g. , 2, 4, 6, 8, 12, 16, 32, or 64 LRP1 ligands). Some or all of the surface branches of the dendrimer can have a LRP1 ligand attached. The linkage between the LRP1 ligand and the dendrimer can be a cleavable linkage (e.g. , a thioester linkage) or a non-cleavable linkage (e.g. , a maleimide linkage). The LRP1 ligand can be attached to the surface branches of the dendrimers via linkers described herein.

In some embodiments, the dendrimeric targeting moiety has the structure:

In other embodiments, the dendrimeric targeting moiety has the structure:



LRP1 Ligands

The conjugates of the invention can feature any dendrimeric targeting moiety that comprises a ligand that interacts with LRP1 , for example, a polypeptide such as Angiopep-1 , Angiopep-2, Angiopep-7, Angiopep-2-4D, reversed Angiopep-2, reversed Angiopep-2 all D, or a fragment or analog thereof. In certain embodiments, the polypeptide may have at least 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or even 100% identity to a polypeptide described herein. The polypeptide may have one or more (e.g. , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15) substitutions relative to one of the sequences described herein. Other modifications are described in greater detail below.

The invention also features fragments of polypeptides (e.g. , a functional fragment) that interact with LRP1. In certain embodiments, the fragments are capable of efficiently being transported to or accumulating in a particular cell type (e.g., liver, eye, lung, kidney, or spleen) or are efficiently transported across the BBB. Truncations of the polypeptide may be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or more amino acids from either the N-terminus of the polypeptide, the C-terminus of the polypeptide, or a combination thereof. Other fragments include sequences where internal portions of the polypeptide are deleted.

Additional polypeptides may be identified by using one of the assays or methods described herein. For example, a candidate LRP1 ligand may be produced by conventional peptide synthesis, conjugated with IDUA, and administered to a laboratory animal. A biologically-active conjugate may be identified, for example, based on its ability to increase survival of an animal model of MPS-I and treated with the conjugate as compared to a control which has not been treated with a conjugate (e.g. , treated with the unconjugated IDUA). Alternatively, a biologically active conjugate may be identified based on its ability to decrease GAG levels in an animal model of MPS-I as compared to a control which has not been treated with a conjugate (e.g. , treated with unconjguated IDUA) or as compared to a healthy control model or a model of a heterozygote carrier of MPS-I. For example, a biologically active polypeptide may be identified based on its location in the parenchyma in an in situ cerebral perfusion assay.

Assays to determine accumulation in other tissues may be performed as well. Labelled conjugates can be administered to an animal, and accumulation in different organs can be measured. For example, a conjugate including a detectable label (e.g. , a near-IR fluorescence spectroscopy label such as Cy5.5) allows live in vivo visualization. Such a conjugate can be administered to an animal, and the presence of the conjugate in an organ can be detected, thus allowing determination of the rate and amount of accumulation of the conjugate in the desired organ. In other embodiments, the conjugate can be labelled with a radioactive isotope (e.g. , ²⁵ l). The conjugate is then administered to an animal. After a period of time, the animal is sacrificed and the organs are extracted. The amount of radioisotope in each organ can then be measured using any means known in the art. By comparing the amount of a labeled candidate conjugate in a particular organ relative to the amount of a labeled control conjugate, the ability of the candidate conjugate to access and accumulate in a particular tissue can be ascertained. Appropriate negative controls include any conjugate known not to be efficiently transported into a particular cell type (e.g. , a conjugate including a peptide related to Angiopep that does not cross the BBB, or any other conjugate).

Modified polypeptides

The polypeptide LRP1 ligands, enzymes, fragments, or analogs used in the invention may have a modified amino acid sequence. In certain embodiments, the modification does not destroy significantly a desired biological activity (e.g. , ability to cross the BBB or enzymatic activity). The modification may reduce (e.g. , by at least 5%, 10%, 20%, 25%, 35%, 50%, 60%, 70%, 75%, 80%, 90%, or 95%), may have no effect, or may increase (e.g. , by at least 5%, 10%, 25%, 50%, 100%, 200%, 500%, or 1000%) the biological activity of the original polypeptide. The modified polypeptide LRP1 ligand, enzyme, fragment, or analog may have or may optimize a characteristic of the polypeptide LRP1 ligand, enzyme, fragment, or analog, such as in vivo stability, bioavailability, toxicity, immunological activity, immunological identity, and conjugation properties.

Modifications include those by natural processes, such as posttranslational processing, or by chemical modification techniques known in the art. Modifications may occur anywhere in a polypeptide including the polypeptide backbone, the amino acid side chains and the amino- or carboxy-terminus. The same type of modification may be present in the same or varying degrees at several sites in a given polypeptide, and a polypeptide may contain more than one type of modification. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslational natural processes or may be made synthetically. Other modifications include pegylation, acetylation, acylation, addition of acetomidomethyl (Acm) group, ADP-ribosylation, alkylation, amidation, biotinylation, carbamoylation, carboxyethylation, esterification, covalent attachment to fiavin, covalent attachment to a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of drug, covalent attachment of a marker (e.g. , fluorescent or radioactive), covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation and ubiquitination.

A modified polypeptide can also include an amino acid insertion, deletion, or substitution, either conservative or non-conservative (e.g. , D-amino acids, desamino acids) in the polypeptide sequence (e.g. , where such changes do not substantially alter the biological activity of the polypeptide). In particular, the addition of one or more cysteine residues to the amino or carboxy terminus of any of the polypeptides of the invention can facilitate conjugation of these polypeptides by, e.g. , disulfide bonding. For example, Angiopep-1 , Angiopep-2, or Angiopep-7 can be modified to include a single cysteine residue at the amino-terminus or a single cysteine residue at the carboxy-terminus. Amino acid substitutions can be conservative (i.e. , wherein a residue is replaced by another of the same general type or group) or non-conservative (i.e. , wherein a residue is replaced by an amino acid of another type). In addition, a non-naturally occurring amino acid can be substituted for a naturally occurring amino acid (i.e. , non-naturally occurring conservative amino acid substitution or a non-naturally occurring non- conservative amino acid substitution).

Polypeptides made synthetically can include substitutions of amino acids not naturally encoded by DNA (e.g. , non-naturally occurring or unnatural amino acid). Examples of non-naturally occurring amino acids include D-amino acids, an amino acid having an acetylaminomethyl group attached to a sulfur atom of a cysteine, a pegylated amino acid, the omega amino acids of the formula

NH ₂ (CH ₂ )nCOOH wherein n is 2-6, neutral nonpolar amino acids, such as sarcosine, t-butyl alanine, t- butyl glycine, N-methyl isoleucine, and norleucine. Phenylglycine may substitute for Trp, Tyr, or Phe; citrulline and methionine sulfoxide are neutral nonpolar, cysteic acid is acidic, and ornithine is basic. Proline may be substituted with hydroxyproline and retain the conformation conferring properties.

Analogs may be generated by substitutional mutagenesis and retain the biological activity of the original polypeptide. Examples of substitutions identified as "conservative substitutions" are shown in Table . If such substitutions result in a change not desired, then other type of substitutions, denominated "exemplary substitutions" in Table 1 , or as further described herein in reference to amino acid classes, are introduced and the products screened.

Substantial modifications in function or immunological identity are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side chain properties:

(1 ) hydrophobic: norleucine, methionine (Met), Alanine (Ala), Valine (Val), Leucine (Leu), Isoleucine (lie), Histidine (His), Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe),

(2) neutral hydrophilic: Cysteine (Cys), Serine (Ser), Threonine (Thr)

(3) acidic/negatively charged: Aspartic acid (Asp), Glutamic acid (Glu)

(4) basic: Asparagine (Asn), Glutamine (Gin), Histidine (His), Lysine (Lys), Arginine (Arg)

(5) residues that influence chain orientation: Glycine (Gly), Proline (Pro);

(6) aromatic: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe), Histidine (His),

(7) polar: Ser, Thr, Asn, Gin

(8) basic positively charged: Arg, Lys, His, and;

(9) charged: Asp, Glu, Arg, Lys, His

Other amino acid substitutions are listed in Table 1.

Table 1 : Amino acid substitutions

Original residue Exemplary substitution(s) Conservative substitution

Ala (A) Val, Leu, lie Val

Arg (R) Lys, Gin, Asn Lys

Asn (N) Gin, His, Lys, Arg Gin

Asp (D) Glu Glu

Cys (C) Ser Ser

Gin (Q) Asn Asn

Glu (E) Asp Asp

Gly (G) Pro Pro Original residue Exemplary substitution(s) Conservative substitution

His (H) Asn, Gin, Lys, Arg Arg

He (1) Leu, Val, Met, Ala, Phe, Norleucine Leu

Leu (L) Norleucine, lie, Val, Met, Ala, Phe lie

Lys (K) Arg, Gin, Asn Arg

Met (M) Leu, Phe, lie Leu

Phe (F) Leu, Val, lie, Ala Leu

Pro (P) Gly Gly

Ser (S) Thr Thr

Thr (T) Ser Ser

Trp (W) Tyr Tyr

Tyr (Y) Trp, Phe, Thr, Ser Phe

Val (V) lie, Leu, Met, Phe, Ala, Norleucine Leu

Polypeptide derivatives and peptidomimetics

In addition to polypeptides consisting of naturally occurring amino acids, peptidomimetics or polypeptide analogs are also encompassed by the present invention and can form the LRP1 ligands or lysosomal enzymes, enzyme fragments, or enzyme analogs used in the conjugates of the invention. Polypeptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template polypeptide. The non-peptide compounds are termed "peptide mimetics" or peptidomimetics. Peptide mimetics that are structurally related to therapeutically useful peptides or polypeptides may be used to produce an equivalent or enhanced therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to the paradigm polypeptide (i.e. , a polypeptide that has a biological or pharmacological activity) such as naturally-occurring receptor-binding polypeptides, but have one or more peptide linkages optionally replaced by linkages such as -CH ₂ NH- - CH ₂ S- -CH ₂ -CH ₂ - -CH=CH- (cis and trans), -CH ₂ SO- -CH(OH)CH ₂ - -COCH ₂ - etc. , by methods well known in the art (Spatola, Peptide Backbone Modifications, Vega Data, 1 :267, 1983; Spatola et al. , Life Sci. 38: 1243-9, 1986; Hudson et al. , Int. J. Pept. Res. 14: 177-85, 1979; and Weinstein, 1983,

Chemistry and Biochemistry, of Amino Acids, Peptides and Proteins, Weinstein eds, Marcel Dekker, New York). Such polypeptide mimetics may have significant advantages over naturally occurring polypeptides including more economical production, greater chemical stability, enhanced pharmacological properties (e.g., half-life, absorption, potency, efficiency), reduced antigenicity, and others.

While the conjugates described herein may efficiently cross the BBB or target particular cell types

(e.g. , those described herein), their effectiveness may be reduced by the presence of proteases.

Likewise, the effectiveness of the lysosomal enzymes, enzyme fragments, or enzyme analogs used in the compounds of the invention may be similarly reduced. Serum proteases have specific substrate requirements, including L-amino acids and peptide bonds for cleavage. Furthermore, exopeptidases, which represent the most prominent component of the protease activity in serum, usually act on the first peptide bond of the polypeptide and require a free N-terminus. In light of this, it is often advantageous to use modified versions of polypeptides. The modified polypeptides retain the structural characteristics of the original L-amino acid polypeptides, but advantageously are not readily susceptible to cleavage by protease and/or exopeptidases.

Systematic substitution of one or more amino acids of a consensus sequence with D-amino acid of the same type (e.g. , an enantiomer; D-lysine in place of L-lysine) may be used to generate more stable polypeptides. Thus, a polypeptide derivative or peptidomimetic as described herein may be all L-, all D-, or mixed D, L polypeptides. The presence of an N-terminal or C-terminal D-amino acid increases the in vivo stability of a polypeptide because peptidases cannot utilize a D-amino acid as a substrate. Reverse- D polypeptides are polypeptides containing D-amino acids, arranged in a reverse sequence relative to a polypeptide containing L-amino acids. Thus, the C-terminal residue of an L-amino acid polypeptide becomes N-terminal for the D-amino acid polypeptide, and so forth. Reverse D-polypeptides retain the same tertiary conformation and therefore the same activity, as the L-amino acid polypeptides, but are more stable to enzymatic degradation in vitro and in vivo, and thus have greater therapeutic efficacy than the original polypeptide. In addition to reverse-D-polypeptides, constrained polypeptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods well known in the art (Rizo ef al., Ann. Rev. Biochem. 61 :387-418, 1992). For example, constrained polypeptides may be generated by adding cysteine residues capable of forming disulfide bridges and, thereby, resulting in a cyclic polypeptide. Cyclic polypeptides have no free N- or C-termini. Accordingly, they are not susceptible to proteolysis by exopeptidases. The amino acid sequences of the polypeptides with N-terminal or C-terminal D-amino acids and of the cyclic polypeptides are usually identical to the sequences of the polypeptides to which they correspond, except for the presence of N- terminal or C-terminal D-amino acid residue, or their circular structure, respectively.

A cyclic derivative containing an intramolecular disulfide bond may be prepared by conventional solid phase synthesis while incorporating suitable S-protected cysteine or homocysteine residues at the positions selected for cyclization such as the amino and carboxy termini. Following completion of the chain assembly, cyclization can be performed either (1 ) by selective removal of the S-protecting group with a consequent on-support oxidation of the corresponding two free SH-functions, to form a S-S bonds, followed by conventional removal of the product from the support and appropriate purification procedure or (2) by removal of the polypeptide from the support along with complete side chain de-protection, followed by oxidation of the free SH-functions in highly dilute aqueous solution.

The cyclic derivative containing an intramolecular amide bond may be prepared by conventional solid phase synthesis while incorporating suitable amino and carboxyl side chain protected amino acid derivatives, at the position selected for cyclization. The cyclic derivatives containing intramolecular -S- alkyl bonds can be prepared by conventional solid phase chemistry while incorporating an amino acid residue with a suitable ami no-protected side chain, and a suitable S-protected cysteine or homocysteine residue at the position selected for cyclization.

Another effective approach to confer resistance to peptidases acting on the N-terminal or C- terminal residues of a polypeptide is to add chemical groups at the polypeptide termini, such that the modified polypeptide is no longer a substrate for the peptidase. One such chemical modification is glycosylation of the polypeptides at either or both termini. Certain chemical modifications, in particular N- terminal glycosylation, have been shown to increase the stability of polypeptides in human serum. Other chemical modifications which enhance serum stability include, but are not limited to, the addition of an N- terminal alkyl group, consisting of a lower alkyl of from one to twenty carbons, such as an acetyl group, and/or the addition of a C-terminal amide or substituted amide group. In particular, the present invention includes modified polypeptides consisting of polypeptides bearing an N-terminal acetyl group and/or a C- terminal amide group.

Also included by the present invention are other types of polypeptide derivatives containing additional chemical moieties not normally part of the polypeptide, provided that the derivative retains the desired functional activity of the polypeptide. Examples of such derivatives include (1 ) N-acyl derivatives of the amino terminal or of another free amino group, wherein the acyl group may be an alkanoyl group (e.g., acetyl, hexanoyl, octanoyl) an aroyl group (e.g. , benzoyl) or a blocking group such as F-moc (fluorenylmethyl-O-CO-); (2) esters of the carboxy terminal or of another free carboxy or hydroxyl group; (3) amide of the carboxy-terminal or of another free carboxyl group produced by reaction with ammonia or with a suitable amine; (4) phosphorylated derivatives; (5) derivatives conjugated to an antibody or other biological ligand and other types of derivatives.

Longer polypeptide sequences which result from the addition of additional amino acid residues to the polypeptides described herein are also encompassed in the present invention. Such longer polypeptide sequences can be expected to have the same biological activity and specificity as the polypeptides described above. While polypeptides having a substantial number of additional amino acids are not excluded, it is recognized that some large polypeptides may assume a configuration that masks the effective sequence, thereby preventing binding to a target (e.g., a member of the LRP receptor family). These derivatives could act as competitive antagonists. Thus, while the present invention encompasses polypeptides or derivatives of the polypeptides described herein having an extension, desirably the extension does not destroy the cell targeting activity or enzymatic activity of the compound.

Other derivatives included in the present invention are dual polypeptides consisting of two of the same, or two different polypeptides, as described herein, covalently linked to one another either directly or through a spacer, such as by a short stretch of alanine residues or by a putative site for proteolysis (e.g., by cathepsin, see e.g. , U.S. Patent No. 5, 126,249 and European Patent No. 495 049). Multimers of the polypeptides described herein consist of a polymer of molecules formed from the same or different polypeptides or derivatives thereof.

The present invention also encompasses polypeptide derivatives that are chimeric or fusion proteins containing a polypeptide described herein, or fragment thereof, linked at its amino- or carboxy- terminal end, or both, to an amino acid sequence of a different protein. Such a chimeric or fusion protein may be produced by recombinant expression of a nucleic acid encoding the protein. For example, a chimeric or fusion protein may contain at least 6 amino acids shared with one of the described polypeptides which desirably results in a chimeric or fusion protein that has an equivalent or greater functional activity.

Assays to identify peptidomimetics

As described above, non-peptidyl compounds generated to replicate the backbone geometry and pharmacophore display (peptidomimetics) of the polypeptides described herein often possess attributes of greater metabolic stability, higher potency, longer duration of action, and better bioavailability.

Peptidomimetics compounds can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the 'one-bead one-compound' library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer, or small molecule libraries of compounds. Examples of methods for the synthesis of molecular libraries are known in the art

Once a polypeptide as described herein is identified, it can be isolated and purified by any number of standard methods including, but not limited to, differential solubility (e.g. , precipitation), centrifugation, chromatography (e.g. , affinity, ion exchange, and size exclusion), or by any other standard techniques used for the purification of peptides, peptidomimetics, or proteins. The functional properties of an identified polypeptide of interest may be evaluated using any functional assay known in the art.

Desirably, assays for evaluating downstream receptor function in intracellular signaling are used (e.g. , cell proliferation).

For example, the peptidomimetics compounds of the present invention may be obtained using the following three-phase process: (1 ) scanning the polypeptides described herein to identify regions of secondary structure necessary for targeting the particular cell types described herein; (2) using conformationally constrained dipeptide surrogates to refine the backbone geometry and provide organic platforms corresponding to these surrogates; and (3) using the best organic platforms to display organic pharmocophores in libraries of candidates designed to mimic the desired activity of the native polypeptide. In more detail the three phases are as follows. In phase 1 , the lead candidate polypeptides are scanned and their structure abridged to identify the requirements for their activity. A series of polypeptide analogs of the original are synthesized. In phase 2, the best polypeptide analogs are investigated using the conformationally constrained dipeptide surrogates, lndolizidin-2-one, indolizidin-9- one and quinolizidinone amino acids (l ² aa, l ⁹ aa and Qaa respectively) are used as platforms for studying backbone geometry of the best peptide candidates. These and related platforms (reviewed in Halab et al., Biopolymers 55: 101 -22, 2000 and Hanessian et al., Tetrahedron 53: 12789-854, 1997) may be introduced at specific regions of the polypeptide to orient the pharmacophores in different directions. Biological evaluation of these analogs identifies improved lead polypeptides that mimic the geometric requirements for activity. In phase 3, the platforms from the most active lead polypeptides are used to display organic surrogates of the pharmacophores responsible for activity of the native peptide. The pharmacophores and scaffolds are combined in a parallel synthesis format. Derivation of polypeptides and the above phases can be accomplished by other means using methods known in the art.

Structure function relationships determined from the polypeptides, polypeptide derivatives, peptidomimetics or other small molecules described herein may be used to refine and prepare analogous molecular structures having similar or better properties. Accordingly, the compounds of the present invention also include molecules that share the structure, polarity, charge characteristics and side chain properties of the polypeptides described herein.

In summary, based on the disclosure herein, those skilled in the art can develop peptides and peptidomimetics screening assays which are useful for identifying compounds for targeting an agent to particular cell types (e.g. , those described herein). The assays of this invention may be developed for low-throughput, high-throughput, or ultra-high throughput screening formats. Assays of the present invention include assays amenable to automation. Linkers

The enzyme having IDUA activity, enzyme fragment, or enzyme analog may be bound to the dendrimeric targeting moiety either directly (e.g. , through a covalent bond such as a peptide bond) or may be bound through a linker. Linkers include chemical linking agents (e.g. , cleavable linkers) and peptides.

In some embodiments, the linker is a chemical linking agent. The lysosomal enzyme (e.g. ,

IDUA), enzyme fragment, or enzyme analog and dendrimeric targeting moiety may be conjugated through sulfhydryl groups, amino groups (amines), and/or carbohydrates or any appropriate reactive group. Homobifunctional and heterobifunctional cross-linkers (conjugation agents) are available from many commercial sources. Regions available for cross-linking may be found on the polypeptides of the present invention. The cross-linker may comprise a flexible arm, e.g. , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15 carbon atoms. Exemplary cross-linkers include BS3 ([Bis(sulfosuccinimidyl)suberate]; BS3 is a homobifunctional N-hydroxysuccinimide ester that targets accessible primary amines), NHS/EDC (N- hydroxysuccinimide and N-ethyl-'(dimethylaminopropyl)carbodimide; NHS/EDC allows for the conjugation of primary amine groups with carboxyl groups), sulfo-EMCS ([N-e-Maleimidocaproic acidjhydrazide; sulfo- EMCS are heterobifunctional reactive groups (maleimide and NHS-ester) that are reactive toward sulfhydryl and amino groups), hydrazide (most proteins contain exposed carbohydrates and hydrazide is a useful reagent for linking carboxyl groups to primary amines), and SATA (N-succinimidyl-S- acetylthioacetate; SATA is reactive towards amines and adds protected sulfhydryls groups).

To form covalent bonds, one can use as a chemically reactive group a wide variety of active carboxyl groups (e.g. , esters) where the hydroxyl moiety is physiologically acceptable at the levels required to modify the peptide. Particular agents include N-hydroxysuccinimide (NHS), N-hydroxy-sulfosuccinimide (sulfo-NHS), maleimide-benzoyl-succinimide (MBS), gamma-maleimido-butyryloxy succinimide ester (GMBS), maleimido propionic acid (MPA) maleimido hexanoic acid (MHA), and maleimido undecanoic acid (MUA).

Primary amines are the principal targets for NHS esters. Accessible a-amine groups present on the N-termini of proteins and the ε-amine of lysine react with NHS esters. An amide bond is formed when the NHS ester conjugation reaction reacts with primary amines releasing N-hydroxysuccinimide. These succinimide containing reactive groups are herein referred to as succinimidyl groups. In certain embodiments of the invention, the functional group on the protein will be a thiol group and the chemically reactive group will be a maleimido-containing group such as gamma-maleimide-butrylamide (GMBA or MPA). Such maleimide containing groups are referred to herein as maleido groups.

The maleimido group is most selective for sulfhydryl groups on peptides when the pH of the reaction mixture is 6.5-7.4. At pH 7.0, the rate of reaction of maleimido groups with sulfhydryls (e.g. , thiol groups on proteins such as serum albumin or IgG) is 1000-fold faster than with amines. Thus, a stable thioether linkage between the maleimido group and the sulfhydryl can be formed.

In other embodiments, the linker includes at least one amino acid (e.g. , a peptide of at least 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 40, or 50 amino acids). In certain embodiments, the linker is a single amino acid (e.g. , any naturally occurring amino acid such as Cys). In other embodiments, a glycine-rich peptide such as a peptide having the sequence [Gly-Gly-Gly-Gly-Ser] _n where n is 1 , 2, 3, 4, 5 or 6 is used, as described in U.S. Patent No. 7,271 , 149. In other embodiments, a serine-rich peptide linker is used, as described in U.S. Patent No. 5,525,491. Serine rich peptide linkers include those of the formula [X-X-X- X-Gly] _y , where up to two of the X are Thr, and the remaining X are Ser, and y is 1 to 5 (e.g. , Ser-Ser-Ser- Ser-Gly, where y is greater than 1 ). In some cases, the linker is a single amino acid (e.g. , any amino acid, such as Gly or Cys). Other linkers include rigid linkers (e.g. , PAPAP and (PT) _n P, where n is 2, 3, 4, 5, 6, or 7) and a-helical linkers (e.g. , A(EAAAK) _n A, where n is 1 , 2, 3, 4, or 5).

Examples of suitable linkers are succinic acid, Lys, Glu, and Asp, or a dipeptide such as Gly-Lys. When the linker is succinic acid, one carboxyl group thereof may form an amide bond with an amino group of the amino acid residue, and the other carboxyl group thereof may, for example, form an amide bond with an amino group of the peptide or substituent. When the linker is Lys, Glu, or Asp, the carboxyl group thereof may form an amide bond with an amino group of the amino acid residue, and the amino group thereof may, for example, form an amide bond with a carboxyl group of the substituent. When Lys is used as the linker, a further linker may be inserted between the ε-amino group of Lys and the substituent. In one particular embodiment, the further linker is succinic acid which, e.g. , forms an amide bond with the ε- amino group of Lys and with an amino group present in the substituent. In one embodiment, the further linker is Glu or Asp (e.g. , which forms an amide bond with the ε-amino group of Lys and another amide bond with a carboxyl group present in the substituent), that is, the substituent is an N ^E -acylated lysine residue.

In certain embodiments, the linker includes the structure:

where n is an integer between 2 and 15 (e.g. , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15). In certain embodiments, the linker is an N-Succinimidyl (acetylthio)acetate (SAT A) linker or a hydrazide linker. The linker may be conjugated to the IDUA or the dendrimeric targeting moiety, through a free amine, a cysteine side chain (e.g. , of Angiopep-2-Cys or Cys-Angiopep-2), or through a glycosylation site.

In some embodiments Click-chemistry linkers

In particular embodiments, the linker is formed by the reaction between a click-chemistry reaction pair. In this embodiment, one of the reactive groups (e.g. , a reactive alkyne) is attached to the enzyme and the other reactive group (e.g. , an azido group) is attached to the dendrimeric targeting moiety. Exemplary reactions and click-chemistry pairs include a Huisgen 1 ,3-dipolar cycloaddition reaction between an alkynyl group and an azido group to form a triazole-containing linker; a Diels-Alder reaction between a diene having a 4π electron system (e.g. , an optionally substituted 1 ,3-unsatu rated compound, such as optionally substituted 1 ,3-butadiene, 1 -methoxy-3-trimethylsilyloxy-1 ,3-butadiene,

cyclopentadiene, cyclohexadiene, or furan) and a dienophile or heterodienophile having a 2π electron system (e.g. , an optionally substituted alkenyl group or an optionally substituted alkynyl group); a ring opening reaction with a nucleophile and a strained heterocyclyl electrophile; a splint ligation reaction with a phosphorothioate group and an iodo group; and a reductive amination reaction with an aldehyde group and an amino group (Kolb et al. , Angew. Chem. Int. Ed. , 40:2004-2021 (2001 ); Van der Eycken et al. , QSAR Comb. Sci. , 26: 1 1 15-1326 (2007)).

In particular embodiments of the invention, the dendrimeric targeting moiety is linked to the enzyme moiety by means of a triazole-containing linker formed by the reaction between a alkynyl group and an azido group click-chemistry pair. In such cases, the azido group may be attached to the polypeptide and the alkynyl group may be attached to the enzyme moiety. Alternatively, the azido group may be attached to the enzyme and the alkynyl group may be attached to the dendrimeric targeting moiety. In certain embodiments, the reaction between an azido group and the alkynyl group is uncatalyzed, and in other embodiments the reaction is catalyzed by a copper(l) catalyst (e.g. , copper(l) iodide), a copper(l l) catalyst in the presence of a reducing agent (e.g. , copper(l l) sulfate or copper( ll) acetate with sodium ascorbate), or a ruthenium-containing catalyst (e.g. , Cp*RuCI(PPh ₃ ) ₂ or

Cp*RuCI(COD)).

Exemplary linkers are those containing monofluorocyclooctyne (MFCO), difluorocyclooctyne (DFCO), cyclooctyne (OCT), dibenzocyclooctyne (DIBO), biarylazacyclooctyne (BARAC),

difluorobenzocyclooctyne (DIFBO), and bicyclo[6.1.0]nonyne (BCN).

In some embodiments, the linker includes the structure:

In other embodiments, the linker includes the structure:

In certain embodiments, the linker includes the structure:

Treatment of MPS-I

The present invention also features methods for the treatment of MPS-I. MPS-I is characterized by cellular accumulation of glycosaminoglycans (GAGs), which results from the inability of the individual to break down these products.

In certain embodiments, treatment is performed on a subject who has been diagnosed with a mutation in the IDUA gene, but does not yet have disease symptoms (e.g. , an infant such as a subject that is 1 month to 12 months old or subject under the age of 2). In other embodiments, treatment is performed on an individual who has at least one MPS-I symptom (e.g. , any of those described herein such as mental decline, reduction in height, enlarged organs, facial features such as flat face, depressed nasal bridge, and bulging forehead, and organ and bone enlargement, respiratory problems, such as obstruction or infection, cardiac complications, moderate mental retardation and learning difficulties, short stature, marked smallness in the jaws, progressive joint stiffness, compressed spinal cord, clouded corneas, hearing loss, heart disease, coarse facial features, umbilical hernia, sleep apnea, glaucoma, retinal degeneration, carpal tunnel syndrome or other nerve compression, stiff joints, claw hands and deformed feet, a short neck, and aortic valve disease, and obstructive airway disease.

Treatment may be performed in a subject of any age, starting from infancy to adulthood.

Subjects may begin treatment, for example, at birth, six months, or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 15, or 18 years of age. Administration and dosage

The present invention also features pharmaceutical compositions that contain a therapeutically effective amount of a compound of the invention. The composition can be formulated for use in a variety of drug delivery systems. One or more physiologically acceptable excipients or carriers can also be included in the composition for proper formulation. Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 22nd ed., 2012.

The pharmaceutical compositions are intended for parenteral, intranasal, topical, oral, or local administration, such as by a transdermal means, for prophylactic and/or therapeutic treatment. The pharmaceutical compositions can be administered parenterally (e.g. , by intravenous, intramuscular, or subcutaneous injection), or by oral ingestion, or by topical application or intraarticular injection at areas affected by the vascular or cancer condition. Additional routes of administration include intravascular, intra-arterial, intratumor, intraperitoneal, intraventricular, intraepidural, as well as nasal, ophthalmic, intrascleral, intraorbital, rectal, topical, or aerosol inhalation administration. Sustained release administration is also specifically included in the invention, by such means as depot injections or erodible implants or components. Thus, the invention provides compositions for parenteral administration that include the above mention agents dissolved or suspended in an acceptable carrier, preferably an aqueous carrier, e.g. , water, buffered water, saline, and PBS. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and detergents. The invention also provides compositions for oral delivery, which may contain inert ingredients such as binders or fillers for the formulation of a tablet, and a capsule. Furthermore, this invention provides compositions for local administration, which may contain inert ingredients such as solvents or emulsifiers for the formulation of a cream, and an ointment.

These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 1 1 , more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents, such as in a sealed package of tablets or capsules. The composition in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment.

The compositions containing an effective amount can be administered for prophylactic or therapeutic treatments. In prophylactic applications, compositions can be administered to a subject diagnosed as having a mutation in the IDUA gene. Compositions of the invention can be administered to the subject (e.g. , a human) in an amount sufficient to delay, reduce, or preferably prevent the onset of the disorder. In therapeutic applications, compositions are administered to a subject (e.g. , a human) already suffering from MPS-I in an amount sufficient to cure or at least partially arrest the symptoms of the disorder and its complications (e.g. , an amount sufficient to reduce GAG levels, such as GAG levels in the brain, to the level present in a heterozygote or a subject with normal enzymatic activity). An amount adequate to accomplish this purpose is defined as a "therapeutically effective amount," an amount of a compound sufficient to substantially improve at least one symptom associated with the disease or a medical condition. For example, in the treatment of a MPS-I, an agent or compound that decreases, prevents, delays, suppresses, or arrests any symptom of the disease or condition would be

therapeutically effective. A therapeutically effective amount of an agent or compound is not required to cure a disease or condition but will provide a treatment for a disease or condition such that the onset of the disease or condition is delayed, hindered, or prevented, or the disease or condition symptoms are ameliorated, or the term of the disease or condition is changed or, for example, is less severe or recovery is accelerated in an individual.

Amounts effective for this use may depend on the severity of the disease or condition and the weight and general state of the subject. Laronidase is recommended for weekly intravenous

administration of 0.58 mg/kg body weight. A conjugate of the invention may, for example, be administered at an equivalent dosage (i.e. , accounting for the additional molecular weight of the transport moiety and linker vs. laronidase) and frequency. The conjugate may be administered at an iduronase equivalent dose, e.g. , 0.01 , 0.05, 0.1 , 0.5, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.75, 1.0, 1.2, 1.25, 1.5, 2.0, 2.5, 3.0, 4.0, or 5 mg/kg montly, every other week, weekly, twice weekly, every other day, daily, or twice daily. The therapeutically effective amount of the compositions of the invention and used in the methods of this invention applied to mammals (e.g. , humans) can be determined by the ordinarily-skilled artisan with consideration of individual differences in age, weight, and the condition of the mammal. Because certain conjugates of the invention exhibit an enhanced ability to cross the BBB and to enter lysosomes, the dosage of the conjugates of the invention can be lower than (e.g. , less than or equal to about 90%, 75%, 50%, 40%, 30%, 20%, 15%, 12%, 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5%, or 0.1 % of) the equivalent dose of required for a therapeutic effect of the unconjugated enzyme. The conjugates of the invention are administered to a subject (e.g. a mammal, such as a human) in an effective amount, which is an amount that produces a desirable result in a treated subject (e.g., reduction of GAG accumulation to levels of a heterozygote or normal subject). Therapeutically effective amounts can also be determined empirically by those of skill in the art.

Single or multiple administrations of the compositions of the invention including an effective amount can be carried out with dose levels and pattern being selected by the treating physician. The dose and administration schedule can be determined and adjusted based on the severity of the disease or condition in the subject, which may be monitored throughout the course of treatment according to the methods commonly practiced by clinicians or those described herein.

The conjugates of the present invention may be used in combination with either conventional methods of treatment or therapy or may be used separately from conventional methods of treatment or therapy.

When the conjugates of this invention are administered in combination therapies with other agents, they may be administered sequentially or concurrently to an individual. Alternatively, pharmaceutical compositions according to the present invention may be comprised of a combination of a conjugate of the present invention in association with a pharmaceutically acceptable excipient, as described herein, and another therapeutic or prophylactic agent known in the art.

EXAMPLES

Example 1. Synthesis of IDUAL17

Exemplary conjugates of the invention were synthesized as shown below:

IDUAL17:

The azido targeting moiety has the structure:

wherein the structure of C is

and n is 1.

IDUA7:

The azido targeting moiety is:

and the average value of n is 2.5. Example 2. Testing of Enzymatic Activity of Conjugates

The enzymatic activity of conjugates was determined in vitro by a fluorometric assay with 4- methylumbelliferyl-a-L-iduronide (4-MUBI) from Glycosynth (Winwick Quay, Warrington, Cheshire, England) as substrate using the unpurified proteins (still in culture media). The substrate was hydrolyzed by IDUA to 4-methylumbelliferone (4-MU), which is detected fluorometrically with a Farrand filter fluorometer using an emission wavelength of 450 nm and an excitation wavelength of 365 nM. A standard curve with known amounts of 4-MU was used for determining the concentration of 4-MU in the assay, which is proportional to the IDUA activity.

It is expected that the activity of the enzyme is preserved in the conjugates and that the fluorometric units should be proportional to the mass of the conjugates added to the substrate.

The enzymatic activity of several conjugates was checked and compared with a commercially available IDUA-10xHis. The enzymatic activity of the conjugates showed similar level to the IDUA-10xHis (Figure 1 A-1 D), demonstrating that the enzyme actvity is preserved after conjugation. The data also indicate that IDUAL17 has surprisingly increased stability as the enzymatic activity post-incubation is sustained at a high level for over 96 hours (Figure 1 C)

In order to determine if the expressed proteins were capable of reducing GAG accumulation in cells, fibroblasts taken from an MPS-I patient were used. MPS-I or healthy human fibroblasts (Coriell Institute) were plated in 6-well dishes at 250,000 cells/well in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and grown at 37°C under 5% C0 ₂ . After 4 days, cells were washed once with phosphate bovine serum (PBS) and once with low sulfate F-12 medium (Invitrogen, catalog # 1 1765-054). One ml of low sulfate F-12 medium containing 10% dialyzed FBS (Sigma, catalog # F0392) and 10 μθί ³⁵ S-sodium sulfate was added to the cells, in the absence or presence of

conjugates. Fibroblasts were incubated at 37°C under 5% C0 ₂ . After 48 h, medium was removed and cells were washed 5 times with PBS. Cells were then lysed in 0.4 ml/well of 1 N NaOH and heated at 60°C for 60 min to solubilize proteins. An aliquot is removed for μΒΟΑ protein assay. Radioactivity is counted with a liquid scintillation counter. The data is expressed as ³⁵ S CPM per μg protein. The results for the conjugates show that the activity of the enzyme was preserved after the conjugation to the dendrimeric targeting moiety. A dose-response was observed with the reduction of GAG in MPS-I fibroblasts to that measured in the healthy fibroblast (Figure 2). The EC ₅₀ of IDUAL17 is 0.04 nM while the EC ₅₀ of unconjugated IDUA is .28 nM.

Example 3. In vitro uptake by MPS-I fibroblasts in presence of M6P and RAP

MPS-I fibroblast cells, as described in previous section, were incubated for 24 h with 2.4 nM of IDUA or conjugate in the presence of an excess of M6P, RAP, or M6P+RAP. As shown in Figure 3, the uptake of both monomeric conjugate (IDUAL7) and native IDUA into MPS-I fibroblasts is mainly M6P receptor-dependent. However, uptake of dendrimeric conjugate (IDUAL17) is predominantly LRP1 receptor-dependent as evidenced by the decrease in enzymatic activity in the presence of the LRP1 inhibitor, RAP, compared with the decrease in enzymatic activity in the presence of free M6P.

Furthermore, as shown in Figures 4A-4E, IDUA17 has enhanced binding to LRP1 compared to monomeric conjugate IDUA7, while the unconjugated enzyme has no binding affinity to LRP1.

Example 4. Enzymatic activity of IDUA17 in MPS-I knockout mice

Protocol

Male hemizygous IDUA gene knock-out mice (supplied by the Laboratory of Dr. Lome Clarke at British Columbia University, Dept of Medical Genetics) aged between 21 -23 weeks were dosed via injection into the caudal vein once a week for 4 weeks. Male wild type animals (23 weeks old) were used in test group 1 as a control.

IDUA activity was measured in homogenates of mice brains prepared from MPS-I knockout mice, at 0, 72, and 96 hours after intravenous injection of IDUAL17. Figure 5A shows that a single injection of IDUAL17 restores the IDUA enzymatic activity in MPS-I knockout mice brain homogenate. Figure 5B shows that a single injection of IDUAL17 also restores IDUA enzymatic activity in the liver of MPS-I knockout mice.

Example 5. Effect of conjugates on GAG accumulation in the brain and liver in hemizygous IDUA knock out mice

Protocol

Male hemizygous IDUA gene knock-out mice (supplied by the Laboratory of Dr. Lome Clarke at British Columbia University, Dept of Medical Genetics) aged between 21 -23 weeks were dosed via injection into the caudal vein once a week for 4 weeks. Male wild type animals (23 weeks old) were used in test group 1 as a control.

As shown in Figures 6A and 6B, IDUAL17 was able to reduce GAG levels in the brain and liver by greater than 66% after 96 hours.

Example 6. Effect of conjugates on GM3 accumulation in the brain in hemizygous IDUA knock out mice

To determine the effect of dendrimeric Angiopep-2-IDUA conjugates on GM3 accumulation in vivo, an animal study can be carried out using the following protocol: Twenty mice are used in this study. The mice are divided into 5 anonymous groups, 4 animals each. The mice are submitted to IDUA replacement treatment by administration with IDUA and An2- IDUA conjugates. The controls use IDUA-KO mice injected with vehicle and wild-type (VVT) mice. Brains are divided on left and right hemisphere. Right hemisphere is frozen, then cut into 15 microns sagittal or coronal sections. GM3 immunocytochemistry is performed using a mouse anti-GM3 monoclonal antibody, clone M2590 (Cosmo Bio C. Ltd. , cat. # NBT-M 101/M 102). This antibody recognizes GM3 and cross-react with Sialylpara globoside (NeuAc) and GM3 Lactone. Optical density (OD) measurement is performed using ImageJ software. The OD values represent a mean ± S. E. D. of GM3 levels evaluated in the brain entorhinal cortex.

Other embodiments

All patents, patent applications, and publications, including U.S. Application Nos. 61 ,660,564, filed June 15, 2012, 61/682,991 , filed August 12, 2012, 61/732, 189, filed November 30, 2012, and International Patent Application Nos. PCT/CA2013/050621 , filed August 12, 2013, and

PCT/CA2013/050453, filed June 14, 2013, mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent, patent application, or publication was specifically and individually indicated to be incorporated by reference.