Alexandria University

Institute of Graduate Studies and Research

Department of Biotechnology
Application of Biotechnology

ENZYME THERAPY

Supervised By
Dr. Amira Mohammed Embaby
Prepared By
Abd El- Halim Mohamed

August 2014
 TABLE OF CONTENTS
Contents Pages
I. Abstract 1
II. Introduction 1
a. Enzyme 1
b. Diseases resulted from enzyme deficiency 2
Accumulation of the substrate 2
Accumulation of normally minor metabolites 3
Deficiency of product 4
c. Metabolic Enzyme Deficiency 4
Autosomal recessive disorder 4
X-linked recessive disorders 5
Autosomal dominant disorders 6
III. The enzyme as drug 7
a. Sources of therapeutic enzymes 7
b. Application of enzyme therapy 11
Digestive enzymes 11
Metabolic enzymes 13
1. Lysosomal storage diseases 14
o Enzyme replacement therapy in type 1 Gaucher disease 15
o Fabry disease 16
o The Mucopolysaccharidoses 19
o Pompe disease 22
o Immunologic response to enzyme replacement therapy 25
o Biodistribution of exogenously administrated enzymes 26
o Principals of enzyme replacement therapy 28
o Remaining challenges 28
Enzymes for the treatment of infectious disease 31
Enzymes for the treatment of cancer 31
IV. References 32
 ABSTRACT
Enzymes have greater affinity, specificity, high catalytic efficiency, and the ability to convert
multiple target molecules to the desired products are the important features that distinguish them
from all other types of drugs. Enzyme therapies are becoming more common in disease
treatment. Therapeutic enzymes can be used medically either isolated or combined with other
therapies for treatment of various diseases like cancer, cystic fibrosis, dermal ulcers,
inflammation, digestive disorders etc. Enzymes as direct pharmaceutical products find numerous
applications.

INTRODUCTION
Deficiency of the enzymes leads to different metabolic disorders which lead to the appearance of
the diseases. The enzyme replacement therapy is emerged to try the correction of those metabolic
disorders and for the treatment of the various diseases due to enzyme deficiency
a. Enzymes:

Enzymes are mostly proteins in nature which are present in the cells of the living organisms.
They act as a biocatalyst that decrease the energy of activation of biochemical reaction so it
increase the rate of the biochemical reaction to give a product. Enzymes are only catalyst in the
biochemical reactions and does not change. Enzymes have unique shape and an active reactive
site that allow them to bind to specific molecules (substrates) to convert them to intermediated
compounds and products. The main advantages of enzymes over other catalyst is their stereo-,
regio, chemo- selectivity and specificity for the substrate. Enzymes are participated in all the
biochemical reactions pathways that happens in the live cells.
The regular intake of the enzymes and enzyme rich foods is a key for healthy body and disease
prevention. Some of the function of the enzymes are regulate the growth of the cell to mature
organism, converting food to energy to satisfy body needs and break down and building up of
some components within the cells.

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 b. Disease Resulted From Enzyme Deficiency:
The symptoms of a disease in patients with inborn errors of metabolism are resulted from
metabolic pathway deviation or disturbance caused by deficiency of catalytic enzymes or
transport protein (Fig 1.)

Figure 1: The primary consequences of inborn errors of metabolism.

The figure shows diagrammatically the various possible mutation-sensitive defects affecting the
compartmentalization and metabolism of Compound A. 1, transporter-mediated movement of A from one
compartment to another; 2, defect in the conversion of B to C; 3, increased conversion of B to D caused by
accumulation of B; 4, defect in the interaction between an apoenzyme and an obligatory cofactor; 5, decreased
feedback inhibition of the conversion of A in to B as a result of deficiency of C; and 6, secondary inhibition of the
conversion of E to F caused by accumulation of D.

Accumulation of the substrate:

Accumulation of the substrate due to defect of an enzyme is a main cause of disease in many
inborn errors of metabolism. However, because the metabolism of the substrate requires the
participation of a number of different enzymes, any of which may be deficient as a result of
mutation, the demonstration of accumulation is diagnostically important only to the extent that it
indicates a class of disorders, not one specific disease. For example mucopolysaccharide
accumulation is important as a screening test for inherited defects in mucopolysaccharide
metabolism. However, the metabolism of the individual mucopolysaccharides involves 10 or
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more genetically distinct lysosomal enzymes, and accumulation of the same compound may
occur as a consequence of deficiency of any one of the enzymes. Specific diagnosis in these
disorders requires the demonstration of the specific enzyme deficiency in appropriate tissues,
such as peripheral blood leukocytes or cultured skin fibroblasts.

Table 1: Some examples of inborn errors of metabolism in which symptoms of disease are the result of substrate
accumulation due to enzyme deficiency.
Disease Metabolic defect Accumulating Main clinical findings
substrate
Tay-Sachs - Hexosaminidase A GM2 ganglioside Cerebral neurodegeneration
disease defciency

OTC defciency ornithine transcarbamoylase Ammonium Acute encephalopathy

defciency
Methylmalonic Methylmalonyl-CoA mutase Methylmalonic acid Metabolic acidosis
academia defciency
PKU Phenylalanine hydroxylase Phenylalanine Progressive mental retardation
defciency
Hepatorenal Fumarylacetoacetase deficiency Fumarylacetoacetate Acute hepatocellular dysfunction,
tyrosinemia and
cirrhosis, rickets
Maleylacetoacetate
Abbreviations: OTC, ornithine transcarbamoylase; PKU, phenylketonuria.

Accumulation of normally minor metabolites:

In some disorders, the main cause of the disease is accumulation of a normally metabolite,
produced in excess by a reaction that is usually of trivial metabolic importance. Cataracts in
patients with untreated galactosemia occur as a result of accumulation of sugar alcohol,
galactitol, a normally minor metabolite of galactose due to deficiency of the following enzymes
galactose 1-phosphate uridyltransferase (GALT) and galactokinase. In another example,
accumulation of the normally minor complex lipid metabolite, psychosine, in the brain of infants
with Krabbe globoid cell leukodystrophy excites a subacute in Xammatory reaction, manifested
by the appearance in the brain of multinucleated giant cells, called globoid cells. It also causes
rapid, severe demyelination, out of proportion to the accumulation of galactocerebroside, the
immediate precursor of the defective enzyme, galactocerebrosidase.

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 Deficiency of product:

Deficiency of the product of a specific reaction is another primary consequence of many

inherited metabolic diseases. The extent to which it contributes to disease depends on the
importance of the product. For example, most of the pathologic consequences of defects of
biosynthesis are traceable to deficiency of the product of the relevant reaction in these cases
substrate accumulation plays little or no role in the development of disease. For example 25-
Hydroxycholecalciferol-1- hydroxylase deficiency in vitamin D metabolism leads to deficiency
of the product 1, 25-Dihydroxycholecalciferol which will lead to rickets disease.
Deficiency of products of reactions is important in two other situations that are common among
the inborn errors of metabolism. One of these could be regarded as the result of a metabolic
steal, a term used to explain the occurrence of myopathy in some patients with glycogen storage
disease due to debrancher enzyme deficiency. It was postulated that increased gluconeogenesis
in patients with the disease causes accelerated muscle protein breakdown as free amino acids are
diverted from protein biosynthesis to gluconeogenesis in an effort to maintain the blood glucose
in the face of impaired glycogen breakdown. Another example of the consequences of a
metabolic steal is the occurrence of hypoglycemia in patients with hereditary defects in fatty acid
oxidation. The over-utilization of glucose and resulting hypoglycemia are a consequence of the
inability to meet energy requirements by fatty acid oxidation because of deWciency of one of the
enzymes involved in the process.

c. Causes of Metabolic Enzyme Deficiency:

Most of the metabolisms disorders are due to enzyme deficiency are inherited. Determination of
the pattern of inheritance of a condition is often helpful in making a diagnosis of genetic disease,
and it provides the foundation for genetic counseling. The most important information required
for establishing the pattern of inheritance is a family history covering at least three generations of
relations. October 2010

Autosomal recessive disorder:

Most of the inherited metabolic diseases recognized today are inherited in the same manner.
Disease expression requires that an individual be homozygous for significant, though not

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necessarily the same, mutations in the same gene. In the overwhelming majority of cases,
homozygosity occurs as a result of inheritance of a mutant gene from each parent, who are both
heterozygous for the defect. Examples of autosomal recessive disorders include cystic fibrosis,
hereditary fructose intolerance, mucopolysaccharidoses except Hunter syndrome and Tay-Sachs
disease.

Fig 2: Autosomal recessive inheritance

X-linked recessive disorders:

they are diseases caused by an error in a single DNA gene. X-linked means the error occurs on
the X chromosome which is the 23rd sex-linked X chromosome. Such diseases are sometimes
called "sex-linked" rather than X-linked. These diseases arise from an error on the X
chromosome, which causes disease only when there is no corresponding paired X chromosome
with a good gene. However, since men are XY a man with the bad gene on the X chromosome
must get the disease, because there is no second X chromosome. Since women are XX, they
usually have a second good X chromosome which suppresses the bad X gene, leaving them
disease-free, but as carriers. Examples of X- linked recessive disorders are glucose 6- phosphate
dehydrogenase deficiency, Fabry disease and Hunter syndrome.

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Fig 3: X- Linked recessive inheritance

Autosomal dominant disorders:

Although autosomal dominant mutations are common causes of genetic disease in humans, they
contribute relatively little to the sum total of inherited metabolic disorders. This is probably
because, with a few exceptions, most inherited metabolic diseases are caused by abnormalities in
enzymes that are not involved in the types of interactions or processes required to produce
dominance. Examples for autosomal dominant disorders are porphyrias

Fig 4: Autosomal dominant inheritance

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 THE ENZYME AS DRUG
The enzymes has many roles in the metabolic pathways in the human body so any deficiency in
the enzymes will leads to metabolic disorders which is associated with the appearance of the
symptoms of a diseases. So recombinant enzymes are used as replacement therapy for the
deficient enzyme to prevent the complication of the disease.
Therapeutic enzymes (digestive and metabolic) can be used medically either alone or in
combination with other therapies for the treatment of various diseases safely. Enzymes as drugs
have two important features, they often bind and interact with their substrate with great affinity
and specificity, and they are catalytic and convert multiple target molecules to the desired
products. These two features make enzymes specific and potent drugs for a wide range of
disorders. The most successful application of enzymes includes the removal of cytotoxic
substances, and treatment of life threatening disorders within the blood circulation.

a. Sources of Therapeutic Enzymes:

Sources of therapeutic enzymes include animal, plant, microbial, bacterial and fungal. Microbial
enzymes are preferred over other sources due to their economic production, their enzyme content
is more predictable and controllable, consistency, ease of process modification, optimization and
reliable supplies of raw material of constant composition, but some of them are incompatible
with the human body due to large molecular size of biological catalyst which prevent their
distribution within the somatic cells and adverse immune response reaction because of injecting
the foreign enzyme protein. the problems could be overcome by the technique of drug targetting
and hide the proteinaceous nature of the enzyme by covalent modification (L- glutaminase
modified by covalent attachment of polyethylene glycol did not show any immunogenic adverse
reaction). Other methods to overcome the immunogenic response are entrapment of the enzymes
within artificial liposome, synthetic microspheres and red blood cell ghost have been found to be
useful. Plant and animal sources contain more harmful material than microbes which include
phenolic compounds (from plants), endogenous enzyme inhibitors and proteases
Development of recombinant DNA technology has made feasible the production of large amount
of therapeutic enzymes which were previously difficult and costly to produce. Protein activity is
often modified by rDNA technology and can be overcome by shuffling the functional domain

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and site directed mutagenesis. this is done to modify activities, regulation and to avoid the
unwanted side effects.

The production of recombinant human enzymes involves three main stages:

1. First stage involves cell culture, growth, and harvest. Before genetic engineering, it was
difficult to purify enough enzyme to treat a single patient. Today, special "special cell
production lines" have been created for large scale manufacturing. these cells are frozen in
a cell bank or storage facility prior to use, and they provide the starter material for the
genetic engineering process. the most commonly used cell production line in genetic
engineering is "Chinese hamster ovary cell (CHO)". to produce a certain enzyme, the gene
for that enzyme is obtained from human DNA and inserted into the CHO cells using a
vector, causing them to express or manufacture the enzyme. Recombinant enzyme
production using mammalian cells offers several advantages over microbial systems. For
example, mammalian cells are able to secrete the protein product, hence, obviating the need
for cell homogenization. Furthermore, they are able to perform post-translational
modifications, like glycosylation, which are necessary for the efficacy of protein drugs
targeted as human therapeutics. However, mammalian cells have significantly slower
growth rates compared to microbes and are much more complex in their nutritional
requirements. To meet the demands of commercial production and regulatory requirements,
bio-manufacturers are faced with the challenge of maximizing productivity whilst
controlling product quality.
2. Second stage involve enzyme purification, because enzymes are proteins. they are digested
if swallowed, making oral administration difficult. As a result, enzyme replacement therapy
must be injected directly into the blood stream and must therefore meet very high purify
standards. To purify the enzyme, unwanted substances are first removed. The purification of
the enzyme is done by concentrate the enzyme by ultra filtration then purify by
chromatography separation. Afterwards the enzyme are ready for formulation.
3. Third stage involve filling. In the last stage of production the manufactured enzyme is filled
into vials using a sterile manufacturing process to prevent product contamination. at this
moment the vial is ready to be sealed, labeled and packaged.

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Table 2: Sources of therapeutic enzymes from natural source
Enzyme Source Use
L-Asparaginase. Pseudomonas acidovorans, Antitumour
Acinetobactor sp
-Galactosidase E. coli Antitumour
Serratiopeptidase Serratia marcescens Antiinflammatory
Superoxide dismutase Mycobacterium sp.,Nocardia sp. Anti-oxidant, Antiinflammatory
Trypsin Pancreas of pigs Treat indigestion, Inflammation,
healing from sports injuries and
surgery.
-Lactamase Staphylococci sp.,Citrobacter Penicillin allergy
freundii,
Serratia marcescens, E. coli
Hyaluronidase Bovine testicular tissue extracts Heart attack
Thrombin Pigs or bovine blood Aid to hemostasis
-Amylase Human saliva, Aspergillus sp. Easy digestion
Urokinase Human urine Blood clots, Anticoagulant
Bromelain Pineapple Anti-inflammatory, healing of burn
wounds on the skin
Maltase Plants, yeast and bacteria, Therapy for Pompes disease
Aspergillis oryzae,
brewerss yeast
Table 3: Human enzymes produced by rDNA technology
Enzyme Source Use
Dornase Produced in Chinese hamster ovary Cystic fibrosis
cells
Rasburicase, a recombinant Urate Derived from a cDNA code from a Hyperuricemia
oxidase enzyme modified Aspergillus flavus strain
and expresssed in a modified yeast
strain of Saccharomyces cerevisiae
-galactosidase Produced in Chinese hamster ovary Fabrys disease
cells & human cells

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 Extract of the DNA segment or RNA for Theraputic enzyme from the
human
1

PCR amplification of the DNA segment of the enzyme

Insert cDNA for the enzyme into a vector

3

Transform the vector into a host

4

over express
5

Harvest, Purify and packaging

6

Fig 5: Steps of recombinant enzyme production.

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 b. Applications of Enzyme Therapy:

Enzyme therapy is used for the treatment of enzyme deficiency and was introduced and brought
into light by Edward Howell to the United States in 1920s. Digestive enzymes are used to digest
food and metabolic enzymes are essential for life. Therapeutic enzymes (digestive and
metabolic) can be used medically either alone or in combination with other therapies for the
treatment of various diseases safely. Enzymes as drugs have two important features: they often
bind and act on their targets with great affinity and specificity and they are catalytic and convert
multiple target molecules to the desired products. These two features make enzymes specific and
potent drugs for a wide range of disorders.

Fig 6: Therapeutic enzymes are used in the treatment of a variety of disorders and diseases.

Digestive enzymes:

Digestive enzymes (mainly pancreatics) break down food and help with the absorption of
nutrients into the blood. Digestive enzymes particularly protease, bromelain and lipase can be
extremely beneficial in reducing symptoms and restoring a balanced life.
Enzymes in living foods are little digestion machines. Every persons life span is directly related
to collapse of their enzyme potential. Digestive and immune system along with liver, kidney, and
pancreas etc. depend upon enzymes. Food components enter the stomach laced with digestive
enzymes (protease, amylase and lipase) produced in the pancreas. Pancreatic enzyme products

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(PEPs) contain the active ingredient pancrelipase, a mixture of the digestive enzymes amylase,
lipase, and protease. These digestive enzymes are used to improve food digestion in patients who
do not produce enough pancreatic enzymes. PEPs have been used to treat patients with various
pancreatic disorders, including cystic fibrosis.
The enzymes cellulase and protease help control Candida overgrowth. Cellulase enzyme breaks
down fiber and it is the only digestive enzyme the body does not make. By adding cellulase to
the diet, this fiber may be removed along with mucus and our body is able to achieve balance.
With a Candida yeast infection, cellulase and protease enzymes eat the Candida yeast. The
enzyme protease has the ability to hydrolyze protein. Protease has been used in clinics all over
the world to break down Candida and prevent its overgrowth.
Lactose is a carbohydrate that is found in dairy products. Some people develop digestive
problems, such as gas, diarrhea and flatulence when they consume food with lactose. Lactase
supplements are used to treat lactose intolerance.
Some enzymes are available in combination. lipase to digest fat, amylase to digest starchy
carbohydrate, and protease to digest protein. In the intestine, pancreatic enzymes help to digest
carbohydrates, proteins and starches of a meal. Digestive enzymes as supplements along with
meal share the workload of bodys own pancreatic enzymes for digestion. Pancreatic enzymes
taken between meals passes directly into blood stream to help the elimination of circulating
immune complexes through kidney. Pancreatic enzyme replacement therapy is currently the
mainstay of treatment for nutrient malabsorption, secondary pancreatic insufficiency, cystic
fibrosis, chronic pancreatitis, pancreatic and periampullary cancer. Enzyme supplementation has
greatly improved the condition of patients in pancreatic exocrine insufficiency such as cystic
fibrosis, chronic pancreatitis, pancreatic and periampullary cancer. Digestive enzymes as
supplements may bring improvement in health problems such as heartburn, indigestion,
Gastroesophageal reflux disease, diarrhoea, constipation, diabetes, bloating, and a host of other
health problems.
Pancreatic enzymes are used as digestive aids. Lipases are responsible for digestion of fats and
deficiency of this enzyme leads to malabsorption of fats and fat-soluble vitamins. Amylases
cause the breakdown of starch molecules into smaller sugars and it is secreted by salivary glands
and the pancreas. Proteases (trypsin, chymotrypsin, carboxypeptidases) convert proteins to
amino acids.

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Supplemental proteolytic enzymes are derived from plant and animal sources. Common
proteases include bromelain from pineapple; papain and chymopapain from papaya; the fungal
protease from the Aspergillus oryzae fungi; and trypsin, chymotrypsin, and pancreatin usually
from porcine (pig) or bovine (cow) origin. A variety of proteases including papain, pepsin are
effective in digesting proteins. Bromelain digest proteins and may be an alternative to help
digestion in people who do not make enough digestive enzymes on their own.
Table 4: Digestive enzyme and their therapeutic use
Enzyme Therapeutic Use
Lactase Lactose intolerance, Helps to consume dairy foods without gas, cramps, bloating, or diarrhea

Pancrelipase Cystic fibrosis , chronic pancreatitis, Enzymes help a person who has cystic fibrosis, digest food by
replacing digestive enzymes that are normally released by the pancreas.
Bromelain Treatment of conditions such as malabsorption and lactose intolerance. Bromelain digest proteins
and help in digestion in people who do notmake enough digestive enzymes on their own.
Lipase Treatment of pancreatic steatorrhea

Metabolic enzymes:

Metabolic enzymes are involved in every process of the human body and help to build structure
and remodel new cells.
Adagen1 (pegadamase bovine), used for the treatment of SCID, represents the first successful
application of an enzyme therapy for an inherited disease. The enzyme ADA cleaves the excess
adenosine present in the circulation of these patients and reduces the toxicity to the immune
system of the elevated adenosine levels. The success of the treatment depends upon the
modification of ADA with PEG. PEG enhances the half-life of the enzyme (originally less than
30 min) and reduces the possibility of immunological reactions due to the bovine origin of the
drug. The studies have shown that the efficient entrapment of native ADA in carrier erythrocytes
also improves substantially the half-life of the enzyme.

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Table 5: Approved enzymes designated as orphan drugs in the USA.
Trade name Generic name Indication
Adagen Pegademase bovine For enzyme replacement therapy for ADA in patients with SCID
Ceredase Alglucerase injection For replacement therapy in patients with Gauchers disease type I
Pulmozyme Dornase To reduce mucous viscosity and enable the clearance of airway
secretions in patients with CF
Fabrazyme Agalsidase beta Treatment of Fabrys disease
Aldurazyme Laronidase Treatment of patients with MPS I
Replaga -Galactosidase A Long-term enzyme replacement therapy for the treatment of Fabrys
disease

1. Lysosomal storage diseases:

Lysosomal storage diseases (LSDs) are a class of more than 50 diseases in which the deficiency
of a single lysosomal enzyme causes the progressive accumulation of undigested
macromolecules in the lysosomes in cells of most tissues which include sphingolipids, glycogen,
mucopolysaccharides, and glycoproteins. If left untreated, accumulation of lysosomal storage
causes progressive lysosomal dysfunction, leading to increased manifestations of disease. This
method of treatment is dependent on the delivery of intravenously delivered enzyme to cell-
surface receptors of the affected tissues. The characterization of the specific metabolic and
genetic defects in these disorders has markedly increased our understanding of lysosomal
biology, including enzyme targeting, intracellular transport, and the complex pathways involved
in the degradation of macromolecules.
Beginning in the early 1970s, ERT pilot clinical studies were undertaken in several LSDs (Fabry,
Gaucher, Pompe, and Sandhoff diseases) by intravenous infusion of the respective normal human
enzyme. In each case, the partially purified enzyme was rapidly cleared from the circulation (t1/2
of about 1020 min), and there was evidence for clearance of the respective accumulated
substrates. These early encouraging studies supported the feasibility of enzyme replacement.
However, they also clearly indicated that the treatment of disorders with primary neuronal
involvement was not feasible by this approach, because the intravenously administered enzymes
did not cross the blood-brain barrier. Thus, ERT for disorders with severe neurologic

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involvement (such as Tay-Sachs, Sandhoff, and type A Niemann-Pick diseases) was not feasible,
and focused their efforts on those without significant neurologic involvement.
the major obstacles confronting successful ERT in LSDs at the time. These included the inability
to produce and purify sufficient quantities of lysosomal enzymes, including specific glycoforms;
lack of the animal model for the experiments and the inability to target exogenously
administered enzymes to specific tissue and cellular sites of pathology, particularly bones,
cartilage, and the central nervous system (CNS).

o Enzyme replacement therapy in type 1 Gaucher disease:

Gaucher disease is caused by the deficient activity of -glucocerebrosidase . The primary cellular
site of pathology in Gaucher disease is the macrophage/ monocyte system, and the bone marrow
and reticuloendothelial organs of affected individuals become infiltrated with lipid-laden foam
cells known as Gaucher cells. Patients develop massive enlargement of the liver and spleen,
pancytopenia, and severe skeletal disease, resulting in bone pain and fractures.
To reverse, or at least halt, the disease progression by using ERT with purified -
glucocerebrosidase from human placentae. Although most lysosomal glycoproteins are targeted
to the lysosome via the mannose-6-phosphate receptormediated trafficking system, -
glucocerebrosidase is not. Therefore, to direct the enzyme to the macrophages, the N-linked
oligosaccharide chains were modified by sequential removal of the sialic acid, -galactosyl, and
-N-acetylglucosaminyl residues, thus exposing terminal mannose residues. This mannose-
terminated form of the enzyme was efficiently recognized by the abundant mannose receptors on
macrophage membranes and was then targeted to macrophage lysosomes for substrate
catabolism. The early results of enzyme replacement using the mannose-terminated enzyme had
encouraging but limited clinical effects, most likely due to the small doses administered. So in
these patients, intravenous infusions of large doses (2.03.0 mg kg1) of the mannose-terminated
enzyme reduced the hepatosplenomegaly, improved hematological values, and led to substantial
improvements in bone density as well as other manifestations.
This demonstration that ERT was safe and well tolerated and most notably, that the enzyme
could reverse years of substrate accumulation in these patients provided the first clinical proof of
concept for ERT in LSDs without primary neurologic involvement.
Initially, the -glucocerebrosidase used for ERT (Ceredase, developed by Genzyme Corporation)
was purified from human placentae by industrial-scale techniques. Later, ERT with the mannose-
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terminated, recombinant human enzyme produced in Chinese hamster ovary (CHO) cells
(Cerezyme , also developed by Genzyme Corporation) was shown to be equally effective.
Because an animal model for Gaucher disease did not exist, investigators experimented with the
dose and dose schedule in patients to reduce the cost of therapy while still retaining therapeutic
effectiveness. Eventually, it became appreciated that the clinical response was dose-dependent
and that the maintenance dose was not significantly different from the dose originally used to
reverse the years of substrate accumulation. Moreover, investigators found that 1.6 mg kg1
(equal to 60 units kg1) every two weeks was more effective (and convenient) for type 1 Gaucher
patients than more frequent administration of a lower dose. Also, the interruption of treatment
resulted in substrate reaccumulation and reversal of the hematopoietic improvements. Because
patients with type 1 Gaucher disease have residual enzymatic activity, the immunologic response
to the normal enzyme was not an issue. Administration of the enzyme in patients with the
neurodegenerative type 2 or 3 Gaucher disease did not improve their neurologic manifestations,
because the macromolecular enzyme could not cross the blood-brain barrier even at high doses.
In addition to the mannose-terminated recombinant human -glucocerebrosidase produced in
CHO cells (imiglucerase, trade name Cerezyme, Genzyme Corporation), two other enzyme
preparations have been recently evaluated in type 1 Gaucher patients: velaglucerase alfa (VPRIV
, Shire HGT), which is produced in human fibrosarcoma cells and was recently FDA approved,
and taliglucerase alfa (Uplyso, Protalix Biotherapeutics), which is produced in carrot cells and
was also recently FDA approved.
In terms of the remaining challenges for Gaucher disease, treatment of the neuronopathic
subtypes and improved treatment of bone disease remain the two most important obstacles. For
the neuronopathic subtypes, it does not appear that high-dose therapy or early intervention will
improve the neurologic disease; therefore, alternative therapies using small molecules that cross
the blood-brain barrier or direct delivery of enzymes to the CNS are needed. For bone disease, it
appears that early intervention may modify the ERT response, and combination therapies that
target secondary storage materials or pathological pathways, or that improve the efficacy of
enzyme delivery, may prove important.
The discoveries that the plasma activity of chitotriosidase and the level of chemokine are
indicators of macrophage activation and disease severity have led to the use of these molecules

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as biomarkers for monitoring therapy, and have demonstrated the importance of biomarkers for
developing and monitoring LSD therapies in general.
The success of ERT in type 1 Gaucher disease stimulated investigators to develop and evaluate
enzyme replacement for other LSDs. These efforts were facilitated by the cloning of the cDNAs
and genes encoding the human lysosomal enzymes, the development of eukaryotic
overexpression systems to produce large quantities of the recombinant glycoprotein enzymes,
and the use of gene-targeting techniques to generate knockout murine models for preclinical
studies of ERT. These advances overcame two of the major early obstacles to ERT: the lack of
sufficient amounts of human enzyme and the need for animal models for preclinical studies.

o Fabry disease:

Fabry disease is an X-linked disorder resulting from the deficient activity of -galactosidase A
(-Gal A) and the progressive lysosomal accumulation of its substrate globotriaosylceramide
(GL-3). In classically affected males, who have no detectable -Gal A activity, GL-3
accumulation in the vascular endothelium causes the major disease manifestations. Clinical onset
in affected boys includes burning sensations in the hands, reddish-purple blemishes on the skin,
cloudiness of the cornea, decreased sweating, fever, gastrointestinal difficulties, impaired arterial
circulation and heart attack. With advancing age, the progressive lysosomal GL-3 accumulation
particularly in the microvasculature leads to renal failure, heart disease, strokes, and premature
demise, typically in the fourth or fifth decade. Males with the later-onset subtype have residual
-Gal A activity and no vascular endothelial involvement. These individuals usually develop
renal failure and/or heart disease in adulthood.
ERT was evaluated for Fabry disease in -Gal A knockout mice, which provided the first
information on the biodistribution, organ uptake, and substrate clearance of an intravenously
administered lysosomal enzyme at different doses. Subsequently, ERT was developed in Fabry
patients using recombinant human -Gal A preparations produced in CHO cells (agalsidase beta,
Fabrazyme, Genzyme Corporation) and in human fibrosarcoma cells (agalsidase alfa, Replagal,
Shire HGT). Several studies comparing the two products specific activity, biochemical
composition, and cell uptake in fibroblasts and knockout Fabry mice have found that the
enzymes have essentially the same specific activities and kinetic properties and similar
glycosylation, although Fabrazyme has more mannose- 6-phosphate and greater sialylation. In
vivo administration of the two enzymes to Fabry mice at the same dose indicated that Fabrazyme
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has greater uptake in the kidney and heart, consistent with its higher mannose- 6-phosphate
content.
The safety and effectiveness of ERT with Fabrazyme have been evaluated by two multicenter,
multinational, randomized, double-blind, placebo-controlled clinical trials involving 58 and 82
patients, respectively. Fabrazyme was shown to clear the accumulated GL-3 in the vascular
endothelium of the kidney, heart, and skin and to normalize the plasma GL-3 level. The phase 4
Fabrazyme clinical trial demonstrated that even patients with advanced disease (serum creatinine
between 1.2 and 3.0 mg%), when treated at 1.0 mg kg1 biweekly, had slower progression than
those in a matched placebo group. The effectiveness of ERT with Fabrazyme in stabilizing renal
disease, improving cardiac involvement, and decreasing the extremity pain and gastrointestinal
manifestations has also been reported in large registries, small cohort studies, and recent expert
reviews.
Following a dose-ranging study from 0.07 to 0.1 mg kg1, which did not show a dose effect,
Replagal was evaluated at 0.2 mg kg1 biweekly in two single-site, randomized, double-blind,
placebo-controlled studies. In the pivotal registration study, which randomized 26 male patients,
pain was the primary endpoint, and the enzymes effect on renal function was evaluated. The
FDA advisory committee did not accept the Replagal data for pain or renal function
improvement. A subsequent study randomized 15 male patients to assess the enzymes effect on
cardiac involvement. In the latter study, left ventricular mass was decreased after six months
compared with that in placebo-matched patients; however, the primary endpoint, reduction in
heart biopsy GL-3 levels, did not achieve significance.
ERT dose in Fabry disease has been the subject of much discussion because the approved
Replagal dose is the lowest of all ERTs for the LSDs 0.2 mg Kg-1 while Fabrazyme dose is 1.0
mg Kg-1 (Table 6).
Recognizing these limitations, investigators recently carried out two clinical studies of the two
drugs, both administered at 0.2 mg kg1 biweekly, which did not reduce left ventricular mass,
glomerular filtration rate, pain, or levels of substrate in plasma or urine; both drugs also raised
enzyme antibodies in affected males. Thus, at the same dose, the drugs had similar effects.
In Fabry disease, most classically affected males who have essentially no enzyme activity raise
IgG antibodies to the infused enzymes, whereas later-onset males and most heterozygotes do not.
In an analysis of more than 700 males and females treated with Fabrazyme (who were not

18 | P a g e
subclassified by classic or later-onset phenotype), 73% of males and 12% of females (68%
overall) developed IgG antibodies. The effect of the antibodies on ERT has been studied by
determining cardiac mass and urinary substrate levels, which indicated that high antibody titers
can impact the effectiveness of substrate clearance. Of note is that antibody-positive patients
treated with Fabrazyme at 1.0 mg kg1 biweekly had persistently decreased urinary GL-3 levels
and decreased heart mass, whereas those treated at 0.2 mg kg1 biweekly with Fabrazyme or
Replagal did not.
A novel approach to avoid raising antibodies against recombinant -Gal A was to modify the
highly homologous human enzyme -Nacetylgalactosaminidase (also known as -Gal B) so that
it would hydrolyze GL-3 and related -Gal A substrates. This enzyme engineering approach
succeeded in creating a sheath enzyme; however, its kinetic properties required large amounts of
infused enzyme to achieve the level of -Gal A effectiveness in the Fabry mouse model.
The importance of early diagnosis and treatment of the LSDs has also been emphasized in Fabry
disease, especially in the phase 4 trial in patients with advanced disease. Early treatment of
classically affected males should begin in childhood when the first symptoms occur (or even
before, for optimal results). GL-3 accumulation particularly in the podocytes, where it was
reduced with 1.0 mg kg1 biweekly but not with 0.2 mg kg1 biweekly that was subsequently
cleared with ERT, suggesting that early intervention may even be preventive.
Recently, renal biopsies from affected boys demonstrated significant GL-3 accumulation
particularly in the podocytes, where it was reduced with 1.0 mg kg1 biweekly but not with 0.2
mg kg1 biweekly that was subsequently cleared with ERT, suggesting that early intervention
may even be preventive.
Another challenge is the fact that some heterozygous females with the classic subtype develop
cardiac and/or renal disease, presumably due to the skewing of random X inactivation. The
difficulty is in predicting which heterozygotes will develop these manifestations, as biomarkers
that reliably predict such individuals have not been identified, and thus continual monitoring of
the heterozygotes is required to detect early signs of renal or cardiac involvement.

o The Mucopolysaccharidoses:

They have been clinically delineated into 7 types. ERTs are available for 4 of these disorders
(MPS I, MPS II, MPS VI and MPS IVA), and are under development for several others. Unique
to the MPSs is the fact that the enzymes are each involved in glycosaminoglycan (GAG)
19 | P a g e
degradation, and therefore the patients present with severe connective tissue disease, particularly
in the skin, trachea, joints and bones. In addition, most MPS disorders have CNS involvement,
with the exception of types IH-S, IS, IVA, and VI.
On the basis of prior animal model studies, pivotal multisite, multinational, randomized, double-
blind, placebo-controlled clinical trials documented the clinical benefit of ERT for MPS I, MPS
II, and MPS VI. ERT for these disorders has provided several useful and important lessons. In
general, these therapies reduce the reticuloendothelial cell storage of GAGs, leading to reduced
organomegaly, increased mobility and breathing, and reduced pain in the treated patients. Joint
mobility is also slightly improved. It is also clear from this experience that the intravenously
administered enzymes do not effectively reach the bone growth plates, articular cartilage, or
CNS (Table 7). The improvements in joint mobility observed in some patients are likely due to
soft tissue changes and reduction in inflammation (see below) rather than delivery of the
enzymes to the joint cartilage.
One unique feature of the MPS diseases is that many of the patients undergoing ERT have also
received hematopoietic stem cell transplants (HSCTs), which until recently were the only
available treatment option for patients with these disorders. For example, bone marrow
transplants for these diseases have been undertaken for more than three decades, and hundreds of
patients have been transplanted. Most of this experience is in MPS I and II, with fewer
transplants in the other MPS types. For severely affected MPS IH patients with CNS
involvement, HSCT remains the treatment of choice because the intravenously administered
enzyme cannot cross the blood-brain barrier. Transplantation has been shown to preserve
intellectual development when performed early in the course of the disease, and is indicated for
MPS IH patients under the age of 2. However, this procedure does carry morbidity and mortality
risks, which have improved over time but are still considerable. Of interest is that ERT is
increasingly being used as an adjuvant treatment before HSCT to improve the pretransplant
condition.
Another important lesson of ERT that has emerged from experiences in the MPS disorders
relates to the treatment of neurologic disease. Animal model studies, including those in MPS VII
mice and MPS I dogs, have suggested that the use of very high doses of intravenous enzymes
very early in life (presymptomatic) could reduce GAG storage in the CNS and partially improve
brain disease. This approach has not been studied in humans, and at present MPS patients are

20 | P a g e
treated only at the time of first clinical diagnosis, with enzyme doses (0.581.0 mg kg1 weekly;
Table 6) that have been shown to improve non-neurologic endpoints but are much lower than
those used in the animal model studies. Therefore, under these conditions the systemically
administered enzymes have not been effective at treating or even stabilizing the CNS
complications of these disorders.
An alternative to high-dose systemic ERT for the CNS component of the LSDs that has been
pioneered in the MPS disorders is intrathecal and/or intraventricular administration of the
enzymes. This has been studied in several MPS animal models and in a very limited number of
MPS patients. For example, a recent animal study showed that intracerebroventricular and
lumbar intrathecal administration of recombinant iduronate-2- sulfatase (the enzyme deficient in
MPS II) in dogs and nonhuman primates results in widespread enzyme distribution in the brain
parenchyma, including in the lysosomes of both neurons and oligodendrocytes. Lumbar
intrathecal administration also resulted in enzyme delivery to the spinal cord, where small
amounts of enzyme were detected after intraventricular administration. Another recent study in
MPS I cats showed that repeated intrathecal injection of recombinant human -L-iduronidase
reduced GAG storage to normal levels in the brain and, most important, showed that the storage
material did not reaccumulate for up to 1 month after the last injection. These results suggested
the potential of intrathecal enzyme dosing every 2 3 months to alleviate GAG storage in the
MPS brain, a finding that has been further supported by other animal model studies as well.
Based on these studies, intrathecal enzyme therapy has also been undertaken in a small number
of MPS IH patients. Although some modest improvements have been reported, the long-term
clinical outcomes and safety of this approach remain to be determined. These early studies have
also highlighted some of the difficulties with repeat administration of enzymes by the intrathecal
route.
The CNS-directed studies have similarly highlighted the importance of appropriate biomarkers to
follow the effectiveness of treatment. For example, analysis of GAG storage in cerebrospinal
fluid has been suggested, as well as measurement of the levels of the heparin cofactor II
thrombin complex.
Another outcome of ERT that has emerged from the early experiences in MPS patients is that
although the systemically administered enzymes are generally useful in improving soft tissues in
the skeletal system of these patients (ligaments, tendons, etc.), they are not effective in the

21 | P a g e
cartilage and bones themselves. Over time, therefore, the soft tissues cannot support the heavy,
dense bones in these individuals, leading to additional bone complications, particularly in the
spine. An unexpected outcome of ongoing ERT in these patients might therefore be a worsening
of certain aspects of their bone diseaseleading, for example, to more surgical intervention to
correct spinal compressions.
This observation has led investigators to more carefully examine the mechanisms of cartilage
and bone disease in MPS, with the goal of identifying additional therapies that could be used in
combination with ERT to alleviate them. For example, it is now clear that GAG storage in MPS
cartilage induces TLR4 signaling and TNF--mediated inflammation. Treatment of MPS animal
models with anti- TNF- antibody therapy significantly reduced articular chondrocyte death and
improved both cartilage histology and growth plate organization. Synovial tissue hyperplasia
characteristic of the MPS diseases was also reduced by anti-TNF- therapy. Most important,
when used in combination with ERT in a rat model of MPS VI, this therapy led to enhanced
bone growth, increased motility, and markedly improved tracheal morphology. This proof of
concept experiment demonstrated the importance of inflammation in MPS bone and joint disease
and the value of anti-inflammatory combination therapies.
Finally, ERT experiences in MPS animal models and patients have also shown that very early
intervention improves the effectiveness of ERT in the bones (and CNS, as mentioned above).
These studies have highlighted the importance of newborn screening for these diseases and the
importance of initiating therapy as soon as possible.

o Pompe disease:

Pompe disease (glycogenosis type II) is an autosomal recessive disorder that results from the
deficient activity of acid -glucosidase and the lysosomal accumulation of glycogen, primarily in
smooth and skeletal muscle throughout the body. The infantile-onset form is characterized by
hypertrophic cardiomyopathy, significant hypotonia, macroglossia, and death in the first year of
life owing to cardiorespiratory failure. In contrast, the later-onset forms (childhood, juvenile,
adult onset) present with progressive muscle weakness with involvement of the respiratory
muscles. These patients can present as early as after the first year of life to as late as the sixth
decade. With disease progression, patients can become wheelchair-bound and ventilator-
dependent.

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In Pompe disease, the challenge for ERT was to clear the accumulated glycogen from muscle
(smooth and skeletal muscle); both types of tissue are hard to reach (Table 7), but the latter is
more difficult, presumably owing to the low abundance of the mannose-6-phosphate receptor in
skeletal muscle. Following preclinical studies in animal models, clinical trials were first
conducted in infantile onset patients with human recombinant acid -glucosidase (alglucosidase
alfa, Myozyme , GenzymeCorporation) produced in CHO cells or in transgenic rabbits. Because
muscle is hard to reach, enzyme doses of 20 40 mg kg1 weekly or biweekly were needed to
overcome the limited biodistribution to muscle cells, particularly the skeletal and respiratory
system muscles (e.g., the diaphragm and external intercostal muscles); in the latter, these doses
improved muscle morphology in both infantile and later onset patients.
ERT in infantile-onset patients has resulted in improved cardiac function and significantly
decreased left ventricular wall thickness and mass. Clinical trials of acid -glucosidase (20 mg
kg1 biweekly) in later-onset patients improved walking distance and stabilized neuromuscular
and pulmonary function. Overall, the response to ERT was generally positive, particularly with
early treatment, and resulted in increased survival and improved motor function; however, the
clinical response in patients has been remarkably variable.
The variable effectiveness of ERT in both subtypes is primarily due to several factors, including
age/stage of disease at ERT start, muscle fiber type, defective autophagy, and immune response
to the infused enzyme. The formation of antibodies is highly dependent on the patients CRIM
status, which in turn depends on the patients specific acid - glucosidaseencoding gene (GAA)
mutations. CRIM-negative patients have no mutant enzyme protein and can raise high titers of
IgG antibodies against the recombinant enzyme, thereby resulting in substrate reaccumulation
and disease progression. CRIM negative infantile-onset patients that develop high antibody titers
have had a poor clinical response to ERT, with the disease continuing to progress to invasive
ventilation or demise. In contrast, most CRIM positive infantile onset patients have low antibody
titers, develop tolerance, and improve with ERT. Importantly, a subset of such CRIM positive
patients also develop high antibody titers against the wild-type enzyme (likely due to the nature
of the underlying mutations) and have an attenuated response to ERT.
To address these challenges, recent studies have attempted to predict the CRIM status of patients
based on Western-blot analyses of cultured fibroblasts and the patientsGAA mutations. Of more
than 240 patients studied, 25% were CRIM negative; most of these patients had nonsense

23 | P a g e
mutations, frameshift mutations, and/or large deletions. Initial genotyping and prediction of the
CRIM status of newly identified patients are important for predicting the efficacy of ERT in
Pompe disease, particularly because recent studies have shown that immunomodulation of
CRIM-negative patients can lead to tolerization if initiated prior to or shortly after the initiation
of ERT. Importantly, CRIM negative patients have been successfully tolerized by a short course
of immunomodulation with rituximab, methotrexate, and intravenous immunoglobulin. In those
who did not tolerize after this regimen, a course of bortezomib did induce tolerance. Further
experience is needed with immunomodulation to induce tolerance in the setting of patients with
high, sustained antibody titers.
Efforts are also under way to develop second-generation recombinant acid - glucosidases (and
other enzymes), either by increasing the mannose-6-phosphate content through neoglycosylation
or by generating a chimeric fusion protein of acid -glucosidase and IGF2, which efficiently
binds the mannose-6-phosphate receptor. A phase 1/2 open-label clinical trial is under way to
evaluate IGF2 fusion enzyme at doses of 5, 10, and 20 mg kg1 biweekly in later-onset patients.
Another intriguing approach under evaluation for Pompe disease that may also be applicable to
other ERTs is the upregulation of the mannose-6-phosphate receptor gene (MPR) to enhance the
number of receptors on cell surfaces for increased enzyme uptake. Studies in double-knockout
mice with a muscle-specific conditional MPR knockout and a ubiquitous GAA knockout have
shown how dependent enzyme uptake is on these receptors. Administration of the selective (2)
agonist (clenbuterol) enhanced MPR expression in skeletal muscle and other tissues, suggesting
that the efficacy of ERT in Pompe disease and other LSDs may be enhanced by this combined
therapy.
Moreover, the recent experience with newborn screening and early ERT resulted in markedly
improved outcomes in Pompe disease. Therefore, newborn diagnosis, rapid prediction of the
CRIM status by genotyping, and early initiation of ERT with immunomodulation in CRIM-
negative patients may overcome some of the challenges in this and other LSDs and improve
therapeutic outcomes.

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 o Immunologic response to enzyme replacement therapy

The absence or presence of the mutant enzyme protein [i.e., cross-reactive immunologic material
(CRIM) negative or positive, respectively] in patients with LSDs primarily determines the
immunologic response to ERT.
In type 1 Gaucher disease, all patients have residual -glucocerebrosidase activity (<10% of
normal), and experience with more than 5,500 treated patients has documented that fewer than
15% of these individuals raise immunoglobulin G (IgG) antibodies against the normal enzyme;
these antibodies have no measurable effect on efficacy (i.e., are non-neutralizing) and rarely
cause infusion-associated reactions. In contrast, the majority of patients with classic Fabry
disease, infantile-onset Pompe disease, type 2 or 3 Gaucher disease, and the severe forms of
MPS I, II, and VI all of whom have essentially no residual enzyme activity develop IgG
antibodies (see Table 6), typically after four to eight infusions. These patients may also
experience infusion-associated reactions including chills, rigors, and/or fevers, which do not
markedly affect efficacy and can be managed conservatively by premedication with non-sedating
antihistamines and antipyretics and by slowing the infusion rate, because these reactions are
directly related to protein load (see Table 6). Importantly, patients who seroconvert decrease
their antibody titers with time and may eventually develop tolerance to the recombinant enzyme.
In rare instances, an LSD patient will raise IgE antibodies and have a life-threatening
anaphylactic reaction. These patients require special treatment to induce tolerance.
In some patients, especially those who are CRIM negative and have high antibody titers, the IgG
antibodies may neutralize a portion of the infused recombinant enzyme activity and/or block the
mannose-6-phosphate moieties, resulting in decreased lysosomal delivery and/or substrate
catabolism. In infantile onset Pompe disease, in which high doses of enzyme are administered
(20 mg kg1 biweekly), CRIM negative and some CRIM positive patients may develop high
antienzyme antibody titers (>1 in 200,000), which may reverse the initial clinical improvement.
The antigen-IgG-antibody complex may be taken up into cellular lysosomes via the Fc receptor,
which will be taken up primarily by macrophages.

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Table 6: Recommended doses, infusion-associated reactions, and antibody formation of human recombinant
enzymes used to treat lysosomal storage disorders
Disease Subtype(s) Recombinant enzyme along Administered dose Proportion of Proportion
with generic and trade name and schedule patients with of
infusion patients
associated with
reactions IgG
antibody
formation
Gaucher Type 1 -Glucocerebrosidase: 1.6 mg kg1 13.8% 15%
disease imiglucerase (Cerezyme) biweekly
-Glucocerebrosidase: 1.6 mg kg1 52% 1.9%
velaglucerase alfa (VPRIV) biweekly
Fabry disease Both classic -Galactosidase A: agalsidase 1.0 mg kg1 50%55% 68%
and later beta (Fabrazyme) biweekly
onset -Galactosidase A: agalsidase 0.2 mg kg1 52% 64%
alfa (Replagal) biweekly
MPS type I Hurler Scheie -L-Iduronidase: laronidase 0.58 mg kg1 weekly 32% 97%
and Scheie (Aldurazyme)
syndromes
MPS type II Both severe Iduronate-2-sulfatase: 0.5 mg kg1 weekly 15% 47%
and idursulfase (Elaprase)
attenuated
MPS type VI N-Acetylgalactosamine-4- 1.0 mg kg1 weekly 54.5% 97%
sulfatase: galsulfase
(Naglazyme)
Pompe Infantile Acid -glucosidase: 20 mg kg1 weekly 51% 95%
disease onset alglucosidase alfa (Myozyme)
Later onset Acid -glucosidase: 20 mg kg1 weekly 5% 100%
(juvenile and alglucosidase alfa
adult) (Lumizyme)

o Biodistribution of exogenously administrated enzymes:

Animal model and clinical studies have revealed organ-specific variations in response to ERT.
The variable organ response is primarily due to the biodistribution of the infused enzymes and

26 | P a g e
the relative density of the lysosomal receptors (e.g., mannose-6-phosphate, Limp2) on different
cell types. Indeed, in the animal models it was found that for most recombinant lysosomal
enzymes the biodistributions following intravenous injection were similar with good distribution
to the reticuloendothelial system and poor uptake by the brain and bones. Other clinically
relevant organs (e.g., the kidneys in Fabry disease, the lungs in Niemann-Pick disease) received
relatively small amounts of enzyme. Notably, in the animal models, the tissue distribution of the
intravenously infused enzymes and the amount and duration of substrate clearance (i.e.,
pharmacodynamics) from target sites of pathology were also dose-dependent. Table 7 lists the
tissue sites of pathology in some human LSDs that are easy or hard to reach based on the
biodistribution and uptake of intravenously administered enzymes in the LSD animal models. As
noted above, for each disease the infused enzymes must be delivered to specific and unique cell
types, which explains why ERT is more effective for some LSDs than for others. For example, in
type 1 Gaucher disease the major pathological cell type is the easily targeted macrophage;
however, treatment must begin early to influence the progressive bone disease. Furthermore,
ERT did not reverse the neurologic manifestations in patients with type 2 or 3 Gaucher disease.
Fabry disease, the major site of pathology is the vascular endothelium, which is readily accessed
by exogenous enzymes, whereas the heart and kidneys take up <1% of the administered enzyme.
In the MPSs, the severe bone and joint abnormalities result from defects in connective tissue
cells (e.g., chondrocytes), which take up little if any of the intravenously administered enzyme.
As noted above, even for diseases with the same enzyme deficiency, certain clinical subtypes
may be more amenable to ERT than otherse.g., type 1 Gaucher disease but not type 2 or 3.
Another example is MPS type I: The severe subtype, Hurler syndrome (MPS IH), results in
early-onset skeletal and neurologic manifestations, whereas the Hurler-Scheie (MPS IH-S) and
Scheie (MPS IS) subtypes have manifestations that are more attenuated, characterized by later
onset and the absence of mental retardation. Thus, the MPS IH-S and IS subtypes are more
amenable to ERT than the MPS IH subtype. Clearly, the effectiveness of ERT in LSDs depends
both on the delivery of sufficient amounts of the administered enzyme to the specific target sites
of pathology and on the reversibility of certain clinical manifestations.

27 | P a g e
Table 7: Easy- and hard-to-reach tissues for in vivo delivery of intravenously administered enzymes
Disease Subtypes Easy to reach Hard to reach
Gaucher disease Type 1 Spleen, liver, bone marrow Bone
Types 2 and 3 Spleen, liver, bone marrow Bone, brain
Fabry disease Both classic and later onset Vascular endothelium Kidney, heart
Mucopolysaccharidoses All Spleen, liver, bone marrow Bone, brain, cartilage

o Principals of enzyme replacement therapy:

As highlighted above, the experience in treating six LSDs have revealed the essential principles
for ERT. The essential principles are as follows:

1. Enzyme biodistribution and lysosomal delivery are receptor-mediated. Enzyme uptake is

dependent on the receptor density on cell membranes (mannose-6-phosphate receptors for
most LSDs, mannose and Limp2 receptors forGaucher disease).Therefore, the enzymes
mannose-6- phosphate content and sialylation must be maximized for optimal lysosomal
uptake for most LSDs. Macromolecular enzymes do not cross the blood-brain barrier.
2. Enzyme delivery and substrate clearance are dose dependent. Adequate doses are required
for delivery to critical sites of pathology, which are disease-specific. Certain tissues are easy
to reach; others are hard to reach and require higher doses (Table 6).
3. Interruption or cessation of ERT leads to substrate reaccumulation and may exacerbate
clinical manifestations.
4. Immune reactions depend on the presence or absence of residual mutant enzyme proteins.
CRIM status may be predicted by genotyping for some diseases, and initial/early
immunomodulation may induce tolerance and optimize therapy.
5. Early treatment improves clinical outcomes and may prevent irreversible disease. Newborn
screening and early intervention offer optimal outcomes.
o Remaining challenges:
The remaining challenges are as follows:
1. Delivery to difficult sites of pathology. New techniques are needed to reach difficult sites
of pathology, such as the bones, cartilage, and brain. These may include direct delivery of
the enzymes (such as intrathecal administration for the brain, which is being evaluated for
MPS) as well as intra-articular administration for the bones (which has been studied in the
MPS animal models). Several laboratories are also developing methods to increase the
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mannose-6-phosphate content of enzymes or create active and stable enzyme fusion proteins
with cell-type-specific targeting sequences. New enzyme formulations are also being
developed as an alternative or supplement to intravenous administration (e.g., aerosols for
the lung and intramuscular injections for the muscles). A clinical trial in mouse was done to
alternative strategies to deliver the lysosomal enzyme - glucuronidase in the enzyme-
deficient mucopolysaccharidosis type VII mouse model. to deliver - glucuronidase. First,
we used a chimeric protein of the insulin-like growth factor II (IGF-II) fused to -
glucuronidase to deliver enzyme via the IGF-II binding site on the bifunctional the insulin-
like growth factor II/ cation-independent mannose 6-phosphate receptor IGF-II/mannose 6-
phosphate receptor. Second, we used the 11-amino-acid human immunodeficiency virus
(HIV) Tat domain fused to - glucuronidase to mediate uptake by absorptive endocytosis.
Interaction with heparan sulfate on the cell surface internalizes and delivers the Tat-tagged
enzyme to the lysosome via plasma membrane recycling. Third, we created a chimeric -
glucuronidase fused to the Fc portion of human immunoglobulin G (IgG) Fc, which was
transported by the neonatal Fc receptor from the maternal circulation across the placenta to
sites of storage in fetal tissues. Finally, periodate treatment was used to eliminate interaction
with carbohydrate receptors, creating an enzyme with increased plasma half-life, resulting in
transport across the bloodbrain barrier and clearance of storage in neurons. These strategies
for delivering lysosomal enzymes could also be used to target non lysosomal proteins or
enzymes identified for bioremediation of other conditions. Clinical trial on Morquio A
mouse disease,N-acetylgalactosamine-6-sulfate sulfatase (GALNS) was bioengineered with
the N-terminus extended by the hexaglutamate sequence (E6) toimprove targeting to bone
(E6-GALNS). blood clearance and tissue distribution was assesed. Next, to assess the
effectiveness of storage clearance and reversal of pathological phenotype, a dose of 250 U/g
of enzyme was given weekly to Morquio A mice (adults: 12 or 24 weeks, newborn: 8
weeks). Sulfatase modifier factor 1 (SUMF1) was co-transfected to activate the enzyme
fully. The E6-GALNS tagged enzyme had markedly prolonged clearance from circulation,
giving over 20 times exposure time in blood, compared to untagged enzyme. The tagged
enzyme was retained longer in bone, with residual enzyme activity demonstrable at 48 hours
after infusion. The pathological findings in adult mice treated with tagged enzyme showed
substantial clearance of the storage materials in bone, bone marrow, and heart valves,

29 | P a g e
 especially after 24 weekly infusions. Mice treated from the newborn period showed marked
reduction of storage materials in tissues investigated. These findings indicate the feasibility
of using tagged enzyme to enhance delivery and pathological effectiveness in Morquio A
mice.
2. Management of immunologic reactions to ERT. In some patients and diseases,
immunologic reactions to the infused enzymes may limit the efficacy of treatment. New
protocols for early immunomodulation therefore need to be evaluated to determine their
safety and long-term effectiveness in tolerizing individuals in order to continue and optimize
therapy. or the enzyme must be modified by covalent attachment of PEG or by
encapsulation in ghost red blood cells/ liposomes to overcome the adverse immune reaction.
3. Identification of appropriate biomarkers that reflect therapeutic effectiveness. For
some organs, clinical response may take many months or years to be recognized. Therefore,
the identification of appropriate biomarkers that enable reliable prediction and/or monitoring
of clinical responses is needed. Proteomics and metabolomics are likely to play important
roles in this area, as is the availability of animal models that can be used to identify and
evaluate the relevance of prospective biomarkers.
4. Evaluation of combinational therapies. It is clear that ERT will not be completely
effective for all organs. Therefore, new combinational approaches need to be evaluated
using drugs that enhance delivery to hard-to-reach tissues as well as drugs that target
alternative and secondary pathological pathways, such as inflammation.
5. Early identification of patients for early therapy. Early intervention in animal models
and patients results in markedly improved clinical responses. However, for most LSDs,
early identification of patients remains a major challenge, particularly prior to the onset of
irreversible organ damage. Newborn screening programs are being implemented that may
overcome this obstacle to early identifications. However, in combination with these
screening efforts, DNA-based and other methods should be developed to predict the disease
subtypes and the likely occurrence of therapeutic response.
6. Reduction in cost and accessibility of therapy. As newborn screening programs are
implemented and more patients are identified preclinically, the questions of when to
implement therapy for each LSD and how to provide accessibility and reimbursement for

30 | P a g e
 therapy will have to be answered. These are likely to be importantchallenges that will need
to be addressed in the upcoming decade.
Enzyme for the treatment of infectious diseases:

Lysozyme has been used as a naturally occurring antibacterial agent in many foods and
consumer products, because of its ability to break carbohydrate chains in the cell wall of
bacteria. Lysozyme has also been shown to possess activity against HIV, as it has RNase A and
urinary RNase U, which selectively degrade viral RNA opening some exciting possibilities for
the treatment of HIV infection. Other naturally occurring antimicrobial agents are the chitinases.
As an element of the cell wall of various pathogenic organisms, including fungi, protozoa and
helminths, chitin is a good target for antimicrobials. The cell walls of Streptococcus pneumonia,
Bacillus anthracis and Clostridium perfringens have been targeted with the use of bacteriophage-
derived lytic enzymes. The use of lytic bacteriophages themselves as a treatment for infections is
also being developed and could prove useful against new drug resistant bacterial strains.

Enzymes for the treatment of cancer:

The field of cancer research has some good examples of the use of enzyme therapeutics. Recent
studies have shown that PEGylated arginine deaminase, an arginine degrading enzyme, can
inhibit human melanoma and hepatocellular carcinomas, which are auxotrophic for arginine
owing to a lack of arginosuccinate synthetase activity.
Another characteristic of the process of oncogenesis is proliferation. It has been shown that the
removal of chondroitin sulfate proteoglycans by chondroitinase AC and, to a lesser extent, by
chondroitinase B, inhibits tumor growth, neovascularization and metastasis.
Antibody-directed enzyme prodrug therapy (ADEPT) illustrates a further application of enzymes
as therapeutic agents in cancer. A monoclonal antibody carries an enzyme specifically to cancer
cells where the enzyme activates a prodrug, destroying cancer cells but not normal cells. This
approach is being utilized to discover and develop a class of cancer therapeutics based on tumor-
targeted enzymes that activate prodrugs. The targeted enzyme prodrug therapy (TEPT) platform,
involving enzymes with antibody-like targeting domains, will also be used in this effort.
One of the side-effects of cancer chemotherapy is hyperuricemia, a build-up of uric acid that
results in gouty arthritis and chronic renal disease. Urate oxidase is able to degrade the poorly
soluble uric acid. Interestingly, the gene for this enzyme is present in humans, but possesses a

31 | P a g e
nonsense codon. Recombinant Rasburicase is safe and effective as a uricolytic agent and the
PEGylated form of the enzyme diminishes its immunogenicity and increases its half-life.

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