Getting started . . .

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(No Transcript)
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Outline
1) Introduction / a few opening comments
Getting started . . .

• 2) Xenotransplantation a glycoengineering
  problem!
• Supplies of organs (e.g, hearts) from human
  donors do not meet demand
• Pigs are a potential ethical source of tissues
  and organs, except for a-Gal
• The a-Gal knock out story . . .

• 3) Treatment of disease (directly)
• Can glycoengineering be done in the body in
  real time to treat disease?
• Sialic acid and cancer will be used to illustrate
  opportunities and pitfalls

• 4) Treatment of disease (indirectly)
• Glycoengineering of recombinant proteins is key
  to their efficacy and safety
• Again, these concepts will be illustrated with
  cancer-based examples

• 5) Metabolic Glycoengineering
• Biosynthetic incorporation of non-natural sugars
  into cellular glycans
• An overview of the technology
• Prospects for biomedical translation

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First What is Carbohydrate Engineering?
From Google
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First What is Carbohydrate Engineering?
For pdfs of the introduction, or any chapter,
email me at kyarema1_at_jhu.edu (or check out
http//uqu.edu.sa/files2/tiny_mce/plugins/filemana
ger/files/4300270/1/2/dk3265fm30.pdf)
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First What is Carbohydrate Engineering?
Basically, in 2005 we didnt really know very
precisely
What about now, in 2013?

• Lets define glycoengineering (a subcategory of
  carbohydrate engineering) as
• (primarily) The manipulation of glycans
• (secondarily) for biomedical purposes

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Blood Group Antigens . . .
. . . (the oldest?) example of glycoengineering
H antigen (O type)
B antigen (B type)
A antigen (A type)
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Blood Group Antigens . . .
. . . an example of genetic/enzyme-based
glycoengineering
ABO gene
produces one of three possible proteins
H antigen (O type)
H antigen (O type)
non-functional protein
1
O allele
B antigen (B type)
2
B allele
a1,3-galactosyltransferase
A antigen (A type)
3
A allele
X
X a1,3-GalNActransferase
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Blood Group Antigens . . .
. . . what is their purpose? (i.e., why did
Mother Nature bother?)
The internet has many suggestions . . .
Personality Traits By Blood Type Find out what
blood type you are and see if it matches your
personality.
The scientific literature also has some clues . .
.
Anthropological surveys suggest that the
geographic and racial distribution of human blood
groups reflects susceptibility of populations
with specific blood types to the plague, cholera,
smallpox, malaria and other infectious diseases.
http//www.ncbi.nlm.nih.gov/pubmed/2506033
A person's blood type may affect their risk for
heart disease, according to a new study that
finds people with blood type A, B or AB were more
likely to develop the disease than those with
type O.
http//www.medicalnewstoday.com/articles/249071.ph
p
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Outline
1) Introduction / a few opening comments

• 2) Xenotransplantation a glycoengineering
  problem!
• Supplies of organs (e.g, hearts) from human
  donors do not meet demand
• Pigs are a potential ethical source of tissues
  and organs, except for a-Gal
• The a-Gal knock out story . . .

Next

• 3) Treatment of disease (directly)
• Can glycoengineering be done in the body in
  real time to treat disease?
• Sialic acid and cancer will be used to illustrate
  opportunities and pitfalls

• 4) Treatment of disease (indirectly)
• Glycoengineering of recombinant proteins is key
  to their efficacy and safety
• Again, these concepts will be illustrated with
  cancer-based examples

• 5) Metabolic Glycoengineering
• Biosynthetic incorporation of non-natural sugars
  into cellular glycans
• An overview of the technology
• Prospects for biomedical translation

10
Cardiovascular Disease USAs 1 Killer
About 600,000 people die of heart disease in the
United States every yearthats 1 in every 4
deaths
http//www.cdc.gov/heartdisease/facts.htm
Question where to get replacements for diseased
and worn out hearts?
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One Option Tissue Engineering
Tissue engineering the creation of new tissues
or organs in the laboratory to replace diseased,
worn out, or injured body parts
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A Second Option Xenotransplants
Baby Fae recipient of a baboon heart (ca. 1984)
Ultimately unsuccessful, spawned a backlash based
(in part) on ethical concerns
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A Second Option Xenotransplants
Xenotransplantation (i.e., transplants from other
species) is being pursued because of a dire
shortage of human donors (and ethical concerns
with using primates)
Pigs seem like a good choice to be organ donors
were already eating them, and theyre quite
similar to us!
The creatures outside looked from pig to man, and
from man to pig, and from pig to man again but
already it was impossible to say which was
which. - George Orwell, Animal Farm
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Xenotransplants Overcoming Hyperacute Rejection
1
What is the cause of hyperacute rejection?
(From Nature Biotechnology, March 2002 Volume 20
Number 3 pp 231 - 232)
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Hyperacute Rejection is Due to a-Gal
The role of a-1,3-Gal in hyperacute and acute
vascular rejection
Hyperacute rejection (HAR) is caused by binding
of large amounts of antibody, consisting
predominantly of anti-a-1,3-Gal, to graft blood
vessels, activating large amounts of complement.
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The a-Gal Epitope is a Trisaccharide
Humans and (other) primates do not make a-Gal and
for that reason avoid HAR (but for ethical
reasons, are not considered to be appropriate
sources for large scale organ harvesting and
transplantation (by contrast 35,000,000 pigs are
already being slaughtered each year in the USA)
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Strategies to Overcome Hyperacute Rejection
Strategy 1. Can soluble aGal protect against
hyperacute rejection?
soluble aGal
But it has not worked out, for several reasons
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Strategies to Overcome Hyperacute Rejection
Strategy 2. Knocking out the a-Gal epitope
aGal
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Strategies to Overcome Hyperacute Rejection
Strategy 2. Knocking out the a-Gal epitope

• Three key technologies were required that were
  falling into place in the 1990s
• Identification of the a1,3-galactosyltransferase
  gene (genetics/bioinformatics)
• homologous recombination of the target genes
  (molecular/cell biology)
• adaptation of nuclear transfer technology to pigs
  (large animal genetics)

The aGal gene was cloned in 1995
Immunogenetics. 199541(2-3)101-5. cDNA sequence
and chromosome localization of pig alpha 1,3
galactosyltransferase. Strahan KM, Gu F, Preece
AF, Gustavsson I, Andersson L, Gustafsson
K. Source Division of Cell and Molecular Biology,
Institute of Child Health, London,
UK. Abstract Human serum contains natural
antibodies (NAb), which can bind to endothelial
cell surface antigens of other mammals. This is
believed to be the major initiating event in the
process of hyperacute rejection of pig to primate
xenografts. Recent work has implicated galactosyl
alpha 1,3 galactosyl beta 1,4 N-acetyl-glucosaminy
l carbohydrate epitopes, on the surface of pig
endothelial cells, as a major target of human
natural antibodies. This epitope is made by a
specific galactosyltransferase (alpha 1,3 GT)
present in pigs but not in higher primates. We
have now cloned and sequenced a full-length pig
alpha 1,3 GT cDNA. The predicted 371 amino acid
protein sequence shares 85 and 76 identity with
previously characterized cattle and mouse alpha
1,3 GT protein sequences, respectively. By using
fluorescence and isotopic in situ hybridization,
the GGTA1 gene was mapped to the region
q2.10-q2.11 of pig chromosome 1, providing
further evidence of homology between the
subterminal region of pig chromosome 1q and human
chromosome 9q, which harbors the locus encoding
the AB0 blood group system as well as a human
pseudogene homologous to the pig GGTA1 gene
http//www.ncbi.nlm.nih.gov/pubmed/7528726
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Strategies to Overcome Hyperacute Rejection
Strategy 2. Knocking out the a-Gal epitope

• Three key technologies were required that were
  falling into place in the 1990s
• Identification of the a1,3-galactosyltransferase
  gene (genetics/bioinformatics)
• homologous recombination of the target genes
  (molecular/cell biology)
• adaptation of nuclear transfer technology to pigs
  (large animal genetics)

Molecular biology techniques were maturing . . .
The aGal gene was knocked out in germ line cells
(from Nature Biotechnology, March 2002 Volume 20
Number 3 pp 231 - 232)
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Strategies to Overcome Hyperacute Rejection
Strategy 2. Knocking out the a-Gal epitope

• Three key technologies were required that were
  falling into place in the 1990s
• Identification of the a1,3-galactosyltransferase
  gene (genetics/bioinformatics)
• homologous recombination of the target genes
  (molecular/cell biology)
• adaptation of nuclear transfer technology to pigs
  (large animal genetics)

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The First a-Gal Knockout Pigs were Born on Xmas
Day, 2002
2
Figure 3 Five a1,3GT gene knockout piglets at 2
weeks of age.
Solution Breeding experiments, expected progeny
/, /-, and -/- at a 121 ratio
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Strategies to Overcome Hyperacute Rejection
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Strategies to Overcome Hyperacute Rejection
Strategy 2. Knocking out the a-Gal epitope
(ca. 2003-2005)
Why/How did the -/- aGT Knock Out Pigs still
Express aGal?
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Strategies to Overcome Hyperacute Rejection
Strategy 2. Knocking out the a-Gal epitope
In Any Event, The Low(er) Residual Levels of
a-Gal were Not a Huge Problem
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Strategies to Overcome Hyperacute Rejection
But theres Still (Much!) More Work to Do
While the presence of the foreign Gal sugar is by
far the major signal for initiating an attack by
the immune system, there are other mediators of
immune rejection at play. Revivicor has also
added a human gene to the pigs to produce a
protein called CD46 that moderates the action of
the immune system. This gene addition strategy,
combined with Gal knock-out and immune
suppression drugs, demonstrated encouraging
results of pig hearts in primates, with survival
and function for up to 8 months. Overcoming
hyperacute rejection is only the first, but
essential, step in Revivicor's comprehensive
approach. . . .
If interested, you can consult the companys
website
http//www.revivicor.com/body_xenotransplantation.
htm
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Outline
1) Introduction / a few opening comments

• 2) Xenotransplantation a glycoengineering
  problem!
• Supplies of organs (e.g, hearts) from human
  donors do not meet demand
• Pigs are a potential ethical source of tissues
  and organs, except for a-Gal
• The a-Gal knock out story . . .

• 3) Treatment of disease (directly)
• Can glycoengineering be done in the body in
  real time to treat disease?
• Sialic acid and cancer will be used to illustrate
  opportunities and pitfalls

Moving on . ..

• 4) Treatment of disease (indirectly)
• Glycoengineering of recombinant proteins is key
  to their efficacy and safety
• Again, these concepts will be illustrated with
  cancer-based examples

• 5) Metabolic Glycoengineering
• Biosynthetic incorporation of non-natural sugars
  into cellular glycans
• An overview of the technology
• Prospects for biomedical translation

28
Sialic Acid is Firmly Linked to Disease (e.g.,
Cancer)
Naturally-occurring cell surface glycans
Exogenous (e.g., dietary) sugars
Neu5Ac is the most common sialic acid in humans
1
Glycosylation pathways
Many tumor-associated carbohydrate antigens
(TACAs) contain sialic acid
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Glycoengineering (Direct) Treatment of Disease
To continue the sialic acid example,
sialosides are strongly associated with
metastatic cancer

• a-2,8-linked sialic acids polysialic acid is
  associated with the initial detachment of a tumor
  cell from a primary tumor and immunoevasion

• a-2,3-linked sialic acids are part of the
  sialyl Lewis X (sLeX) epitope involved in
  tethering and rolling during extravasation of a
  cancer cell from the blood into the underlying
  tissue

• a-2,6-linked sialic acids are involved in
  integrin-mediated migration and attachment of
  tumor cells to the extracellular matrix

Question, can these TACAs be knocked out in
cancer cells?
Dove, A. (2001) The bittersweet promise of
glycobiology. Nat Biotechnol 19, 913-917.
Fuster, M. M., and Esko, J. D. (2005) The sweet
and sour of cancer glycans as novel therapeutic
targets. Nat Rev Cancer 5, 526-542.
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How are Tumor-associated Carbohydrate Antigens
Regulated?
The conventional wisdom is through
sialyltransferases (and sialidases)
CMP-Neu5Ac has been presumed to NOT be regulatory
31
The First Step Identification of the Relevant
Gene/Enzyme
CMP-Neu5Ac has been presumed to NOT be regulatory
Sometimes researchers get lucky and identify an
exact gene / enzyme linked to cancer
CMP-Neu5Ac
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The First Step Identification of the Relevant
Gene/Enzyme
Two examples
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The First Step Identification of the Relevant
Gene/Enzyme
Uh Oh - Trouble is afoot!
An elevation of b-galactoside a-2,6-sialyltransfer
ase (ST6Gal.I) enzyme activity and an increased
a-2,6-sialylation of cell membranes are among the
most prominent glycosylation changes associated
with colon cancer both modifications correlate
with a worse prognosis.
Dall'Olio, F., Chiricolo, M., Mariani, E., and
Facchini, A. (2001) Biosynthesis of the
cancer-related sialyl-a2,6-lactosaminyl epitope
in colon cancer cell lines expressing
b-galactoside a2,6-sialyltransferase under a
constitutive promoter. Eur J Biochem 268,
5876-5884.
we have frequently observed a discrepancy
between the ST6Gal.I level within a colon cancer
sample or cell line and the respective level of
reactivity with the a-2,6-sialyl-specific lectin
from Sambucus nigra (SNA)
One reason for such discrepancies lies in the
complex, non-template-based biosynthetic routes
for glycans
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The First Step Identification of the Relevant
Gene/Enzyme
Towards overcoming these discrepancies . . . ..
http//www.ncbi.nlm.nih.gov/pubmed/23326219 http/
/www.ncbi.nlm.nih.gov/pubmed/19506293
One reason for such discrepancies lies in the
complex, non-template-based biosynthetic routes
for glycans
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The Next Step Carrying Out the
Glycoengineering
Lets assume that a candidate gene / enzyme has
been IDed!
More specifically, the level of ST6Gal.I enzyme
activity only partially correlates with the mRNA
level
Dall'Olio, F., Chiricolo, M., Mariani, E., and
Facchini, A. (2001) Biosynthesis of the
cancer-related sialyl-a2,6-lactosaminyl epitope
in colon cancer cell lines expressing
b-galactoside a2,6-sialyltransferase under a
constitutive promoter. Eur J Biochem 268,
5876-5884.
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The Next Step Carrying Out the
Glycoengineering
Lets assume that a candidate gene / enzyme has
been IDed!
The challenge (in general) in the development of
glycosyltransferase inhibitors is to achieve
specificity for one of many very similar enzymes
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Revisiting/Summarizing (Direct) Treatment of
Disease
First, is this even possible?
Dove, A. (2001) The bittersweet promise of
glycobiology. Nat Biotechnol 19, 913-917.
Fuster, M. M., and Esko, J. D. (2005) The sweet
and sour of cancer glycans as novel therapeutic
targets. Nat Rev Cancer 5, 526-542.
Not really, not today (at least for complex
disease like cancer)

• Identification of biomarkers and candidate
  genes / enzymes remains challenging

• Intervention remains equally if not more
  difficult

But hopefully this information is inspiring, not
discouraging!
38
Outline
1) Introduction / a few opening comments

• 2) Xenotransplantation a glycoengineering
  problem!
• Supplies of organs (e.g, hearts) from human
  donors do not meet demand
• Pigs are a potential ethical source of tissues
  and organs, except for a-Gal
• The a-Gal knock out story . . .

• 3) Treatment of disease (directly)
• Can glycoengineering be done in the body in
  real time to treat disease?
• Sialic acid and cancer will be used to illustrate
  opportunities and pitfalls

• 4) Treatment of disease (indirectly)
• Glycoengineering of recombinant proteins is key
  to their efficacy and safety
• Again, these concepts will be illustrated with
  cancer-based examples

Moving on, and on . ..

• 5) Metabolic Glycoengineering
• Biosynthetic incorporation of non-natural sugars
  into cellular glycans
• An overview of the technology
• Prospects for biomedical translation

39
Glycoengineering (Indirect) Treatment of Cancer
Antibody-dependent cellular cytotoxicity (ADCC)
is an emerging cancer treatment. During ADCC,
antibodies bound to tumor cells recruit innate
immune effector cells that express cellular
receptors (Fc receptors FcRs) specific for the
constant region of the antibody, thereby
triggering phagocytosis and the release of
inflammatory mediators and cytotoxic substances
Nimmerjahn, F., and Ravetch, J. V. (2007)
Antibodies, Fc receptors and cancer. Curr Opin
Immunol 19, 239-245.
 Optimizing antibodyFcR interactions. An
important strategy to obtain a stronger
anti-tumor ADCC reaction is to optimize the
interaction of the antibody Fc-portion with
activating FcRs. This can be achieved by blocking
the inhibitory Fc?RIIB in vivo with monoclonal
antibodies, or by modifying the primary amino
acid sequence (amino acid AA modifications) or
the sugar moiety of the antibody to obtain
selective or enhanced binding to activating FcRs.
40
Glycoengineering Treatment of Autoimmune Disease
Bad for ADCC
Intravenous immunoglobulin (IVIg) therapy is used
to treat a wide range of autoimmune conditions
and consists of pooled immunoglobulin G (IgG)
from healthy donors. The immunosuppressive
effects of IVIg are, in part, attributed to
terminal a2,6-linked sialic acid residues on the
N-linked glycans of the IgG Fc (fragment
crystallizable) domain.
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mAb Glycoengineering But first, Do No Harm
Bad for ADCC
Required for IVIg
A Southern Mystery (from The Scientist, July
1, 2008) In 2004, strange things were happening
when people living in the Southern United States
received Erbitux (aka Cetuximab), an (mAb)
anticancer drug. After Erbitux was approved, the
first three patients that oncologist Bert O'Neil
treated at the University of North Carolina,
Chapel Hill, had severe anaphylactic reactions.
One fell out of their chair," passing out as
blood pressure plummeted. "It alarmed us. "I
was quite upset," says research oncologist
Christine Chung, when her patient with head and
neck cancer had a severe reaction to the drug.
"This was a young man and a last ditch effort" to
gain a little more time for this patient . . . .
The affected patients had IgE antibodies against
galactose-a-1,3-galactose (a-Gal!), which
triggered anaphylaxis when they were given the
drug.
N Engl J Med 2008 3581109-1117 DOI
10.1056/NEJMoa074943
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Glycoengineering But first, Do No Harm
What happened? (in more detail)
Unlike most other monoclonal antibodies,
cetuximab is produced in the mouse cell line
SP2/0, which expresses the gene for
a-1,3-galactosyltransferase. Evidence was
obtained that pre-existing IgE antibodies
specific for a-Gal can cause severe infusion
reactions.
The solution use a safe cell line for mAb
production
A variant of cetuximab, CHO-C225, which is
produced in Chinese hamster ovary (CHO) cell
lines that do not produce a-1,3-galactosyltransfer
ase and, for this reason, has a pattern of
glycosylation that differs from that of cetuximab
was found to be safe to administer to patients
with IgE antibodies against a-Gal.
The affected patients had IgE antibodies against
galactose-a-1,3-galactose (a-Gal!), which
triggered anaphylaxis when they were given the
drug.
70 of recombinant proteins worth 30 billion
are currently made in CHO cells.
http//www.aiche.org/sites/default/files/docs/page
s/CHO.pdf
N Engl J Med 2008 3581109-1117 DOI
10.1056/NEJMoa074943
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CHO Cells A Workhorse Platform for
Glycoengineering
For example, Pam Stanleys lab has developed a
library of CHO mutants allowing desired
glycoforms to be dialed in (or out . .. )
Patnaik Stanley (Methods in Enzymology,
2006) http//www.sciencedirect.com/science/articl
e/pii/S0076687906160115
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Carbohydrate Engineering Do glycan arrays count?
The Technology
Campbell, C. T., Gulley, J. L., Oyelaran, O.,
Hodge, J. W., Schlom, J., and Gildersleeve, J. C.
(2013) Serum antibodies to blood group A predict
survival on PROSTAC-VF. Clin Cancer Res Published
Online, January 29, 2013, doi10.1158/1078-0432.CC
R-1112-2478.
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To Summarize What Weve Learned about mAb-based
Therapies
Outline
Both safety . . .
. . .and efficacy . .. .
. . . are influenced by pre-existing,
glycan-recognizing antibodies
46
Outline
1) Introduction / a few opening comments

• 2) Xenotransplantation a glycoengineering
  problem!
• Supplies of organs (e.g, hearts) from human
  donors do not meet demand
• Pigs are a potential ethical source of tissues
  and organs, except for a-Gal
• The a-Gal knock out story . . .

• 3) Treatment of disease (directly)
• Can glycoengineering be done in the body in
  real time to treat disease?
• Sialic acid and cancer will be used to illustrate
  opportunities and pitfalls

• 4) Treatment of disease (indirectly)
• Glycoengineering of recombinant proteins is key
  to their efficacy and safety
• Again, these concepts will be illustrated with
  cancer-based examples

• 5) Metabolic Glycoengineering
• Biosynthetic incorporation of non-natural sugars
  into cellular glycans
• An overview of the technology
• Prospects for biomedical translation

. . . And finally!
47
Shifting Gears Its Not All About the
Glycosyltransferases
Outline
In the past (up to the present day, really) a
widespread / working assumption has been that
glycan structures are controlled at the level of
glycosyltransferases.
48
Metabolic Glycoengineering Elongating the
NAcyl Group
Naturally-occurring cell surface oligosaccharides
Exogenous (e.g., dietary) sugars
1
Glycosylation pathways
ManNAc
Kayser et al, Journal of Biological Chemistry,
1992
49
Metabolic Glycoengineering Why?
Naturally-occurring cell surface oligosaccharides
Exogenous (e.g., dietary) sugars
1
Glycosylation pathways
Keppler et al, Glycobiology 2001
50
On the Topic of Viruses the Human Microbiome
Project . .
51
Installing New Chemical Reactivity onto the Cell
Surface
Naturally-occurring cell surface oligosaccharides
Exogenous (e.g., dietary) sugars
1
Glycosylation pathways
52
Click Chemistry 1,530,000 Google Entries . . .
That was in 2007 17,300,000 in 2009 32,200,000
in 2010 539,000,000 in 2013
53
This Technology has been Commercialized as a
Glycomics Tool
Sialic acid pathway
Ac4ManNAz
It works best when the cells/animals can be
sacrificed (i.e., when they are dead)
(the copper is somewhat toxic, this problem is
solved on the next slide)
54
New Bio-orthogonal Chemistries can be used In Vivo
Sialic acid pathway
Ac4ManNAz
55
Additional Pathways besides Sialic Acid can be
Targeted
In addition to cell surface sialic acid,
metabolic glycoengineering now can target cell
surface GalNAc and fucose (GlcNAc analogs mainly
label intracellular O-GlcNAc )
56
Expanding the Repertoire of Bio-orthogonal
Chemistries
(A) The ketone is the first example of an
bio-orthogonal chemical functional group
installed in the glycocalyx
(B) and (C) Either click functional group
(azides, B or alkynes, C) can be installed in the
glycocalyx
(D) and (E) Photoactivated functional groups can
be installed in the glycocalyx
(F) Thiols can be incorporated into an unusual
cellular locale, the glycocalyx
contact me for information on our labs efforts
to use sialic acid-displayed thiols for tissue
engineering
Du et al, Glycobiology, 2009
57
Metabolic Glycoengineering A Chemistry-driven
Field (1)
Almaraz et al, Ann Biomed Eng, 2012
58
Metabolic Glycoengineering A Chemistry-driven
Field (2)
NEW!
Previous slide
Previous slide
NEW!
Du et al, Glycobiology, 2009
Aich et al, Glycoscience, 2008
59
Metabolic Glycoengineering A Failed
Biomedical Field?
Two mid-90s developments have not progressed to
the clinic
1) Anti-viral potential of ManNAc analogs (as
previously mentioned)
2) Their anti-cancer potential is similarly
unrealized
60
Challenges facing Clinical Translation of
Metabolic Glycoengineering

1. At the systems level, sugars are not drug-like
   / druggable

• poor oral availability
• rapid serum clearance
• rapid in vivo degradation
• (etc)

1. At the cell level, ManNAc analogs are used very
   inefficiently

107 out
1013 in
61
Re-designing Monosaccharide Analogs to be
Druggable
Analog re-design
(1) increased metabolic efficiency
(2) uncovered biological activities not directly
associated with glycosylation (or the SCFA . . .
.)
ManNAc
62
The Hexosamine Scaffold A Template for Drug
Design ?
Potentially, any of gt 105 permutations affects
biological activity Journal of Medicinal
Chemistry, 2009
Many More!
(cancer)
(Osteoarthritis (OA))

• Applications
• Tissue engineering / manipulation of stem cells
  (our group)
• In vivo imaging

• Diseases
• Cancer (our group)
• Osteoarthritis (collaboration w/ the Elisseeff
  group)
• Muscle disorders HIBM (collaboration with
  Huizing group, NIH)
• - Duchene's
  (collaboration with Varghese group, UCSD)

• Approaches
• New delivery options (e.g., nanoparticles and
  micelles/liposomes)
• Continued exploration of analog SAR
• Investigation of mechanism

63
Outline
1) Introduction / a few opening comments

• 2) Xenotransplantation a glycoengineering
  problem!
• Supplies of organs (e.g, hearts) from human
  donors do not meet demand
• Pigs are a potential ethical source of tissues
  and organs, except for a-Gal
• The a-Gal knock out story . . .

• 3) Treatment of disease (directly)
• Can glycoengineering be done in the body in
  real time to treat disease?
• Sialic acid and cancer will be used to illustrate
  opportunities and pitfalls

• 4) Treatment of disease (indirectly)
• Glycoengineering of recombinant proteins is key
  to their efficacy and safety
• Again, these concepts will be illustrated with
  cancer-based examples

• 5) Metabolic Glycoengineering
• Biosynthetic incorporation of non-natural sugars
  into cellular glycans
• An overview of the technology
• Prospects for biomedical translation

. . . And finally ALL DONE!
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