RTL1 produces a sense mRNA that is targeted for degradation by antisense microRNAs transcribed from the sense strand. Translation of the mRNA produces a retrotransposon-derived protein that is implicated in placental development. The sense and antisense transcripts are oppositely imprinted: sense mRNAs are expressed from the paternally-derived chromosome, antisense microRNAs from the maternally-derived chromosome.Transcription of the antisense strand of RTL1 locus, miR-431 and the rodent-specific miR-434, are derived from within tandem repeats. We present an evolutionary model for the establishment of a new self-targeting microRNA derived from within a tandem repeat that inhibits production of RTL1 protein when maternally-derived in heterozygotes but not when paternally-derived.Two microRNAs at the RTL1 alleles that possesses many of the features of a greenbeard effect. This interaction is evolutionary stable, unlike a typical greenbeard effect, because of the necessary complementarity between microRNAs and mRNA transcribed from opposite strands of the same double helix. We conjecture that microRNAs and mRNA cooperate to reduce demands on mothers when an allele is paired with itself in homozygous offspring.The interaction of sense and antisense transcripts can be interpreted as a form of communication between maternally-derived and paternally-derived This article was reviewed by Eugene Berezikov and Bernard Crespi. Sheep-breeders were unable to fix this desirable trait within a herd because the only animals to express the trait inherited the underlying mutation from their father but not their mother (+/CLPG). The reciprocal heterozygotes who inherited the mutation from their mother (CLPG/+) had rumps of normal proportions as did sheep who inherited the mutation from both parents (CLPG/CLPG) or neither parent (+/+) [From his experiments on hybridization in peas, Gregor Mendel concluded that “it is perfectly immaterial whether the dominant character belong to the seed-bearer or to the pollen-bearer; the form of the hybrid remains identical in both cases” . This idnt (+/+) . The difcallipyge region of ovine chromosome 18 and orthologous regions of human chromosome 14 and mouse chromosome 12 were subsequently found to be the home of a major cluster of imprinted genes [Retrotransposon Gag like 1 (RTL1), is derived from a sushi-ishi-like retroelement [RTL1 is transcribed from the antisense strand of the DNA double helix to produce a protein-coding ‘sense’ mRNA whereas the maternal copy is transcribed from the sense strand to produce multiple ‘antisense’ microRNAs [RTL1 coding sequence and promote degradation of RTL1 mRNA [RTL1 sequence is thus constrained by dual roles as the progenitor of a paternally-expressed mRNA and maternally-expressed microRNAs.The ed genes  includined genes –9. One goelement , 11 thatoelement . The paticroRNAs , 14. In icroRNAs , 16. TheTL1 mRNA –19. The Rtl1 mRNA is expressed in endothelial cells of the labyrinthine zone of the murine placenta. Deletion of the coding sequence results in complete suppression of Rtl1 mRNA when inherited from fathers  but three-fold increased Rtl1 mRNA when inherited from mothers . The latter effect is a consequence of the absence of the maternally-expressed antisense microRNAs that target paternally-expressed Rtl1 mRNA [tl1 mRNA . Both kntl1 mRNA . Mice wiRTL1 causes muscular hypertrophy in mice, suggesting a role of the gene in the callipyge phenotype of sheep [RTL1 is the major cause of post-implantation failure of cloned pigs [RTL1-associated miRNAs, both pro-oncogenic and anti-oncogenic, is beyond the scope of this paper.Overexpression of of sheep , inducesof sheep , and proof sheep . Non-expned pigs . A critiX = xm + xp, where xm was the level of expression of the maternally-derived allele and xp the level of expression of the paternally-derived allele. An allele’s ‘strategy’ in these two roles was represented by the two-element vector {xm, xp}. The models found maternal silence {0, X} to be the unbeatable strategy at loci where higher values of X were favored when paternally-derived and paternal silence {X, 0} to be the unbeatable strategy at loci where higher values of X are favored when maternally-derived.The kinship theory of genomic imprinting was developed to explain the origin and maintenance of imprinted gene expression in prenatal and postnatal life , 27. SimInsulin-like growth factor 2 (Igf2), a paternally-expressed growth-enhancer, and Insulin-like growth factor 2 receptor (Igf2r), a maternally-expressed growth-inhibitor that targets IGF-II to lysosomes for degradation [CDKN1C, a paternally-silent growth-inhibitor [IGF2, the aforementioned maternally-silent growth-enhancer [In the context of maternal–fetal relations and placental development, the theory predicts that PEGs will increase fetal demands on mothers and MEGs will have opposing effects . The claradation . A singlradation . Similarnhibitor  whereas enhancer .RTL1 mRNA and maternally-expressed microRNAs on levels of RTL1 protein have been interpreted as consistent with the kinship theory [Rtl1 is compatible with RTL1 protein functioning as a fetal demand enhancer and the increase of Rtl1 mRNA when the imprinted microRNAs are knocked-out is compatible with the microRNAs acting as demand inhibitors [Rtl1 mRNA.The opposite effects of paternally-expressed p theory . Moreovehibitors , 21. TheRTL1 support the kinship theory is premature because the details of this system violate a key assumption of the mathematical models. This was the assumption that xm and xp were fixed properties of an allele and therefore independent of the identity of the other allele at the locus in a diploid individual. An RTL1 allele is transcribed both as a PEG (mRNA) and as MEGs (microRNAs) that inhibit the PEG. Therefore, the level of expression of the paternal allele may depend on the identity of the maternal allele. At such a locus, an allele’s strategy could be thought of as a three-element vector {xm, xp, xq} where xm and xp are the levels of expression when maternally-derived and paternally-derived in heterozygotes, and xq is the level of expression in homozygotes.The conclusion that the phenotypic effects of Haig , 35 madeClassic formulations of greenbeard effects invoked a triad of trait, recognition, and response: an actor with the trait recognized the same trait in another individual and responded altruistically , 37. Suctrait that is recognized by the microRNA’s target and that informs the targeted sequence of its similarity to the sequence from which the microRNA was transcribed. Regulation of the targeted sequence by the microRNA can be considered the response of the target to this information. If the microRNA acts on an mRNA transcribed from the other allele at its locus, then regulation of the mRNA by the microRNA can be conceptualized as a response to self-recognition. ‘Self-targeting’ microRNAs thus combine all three aspects of the greenbeard triad in a single nucleic acid sequence.A microRNA can be considered a RTL1 locus meets these criteria for ‘self-recognition’ and thus provides a test-case for disentangling the evolutionary complexities of such systems. The current paper investigates the evolutionary history of microRNAs at the RTL1 locus and then develops an evolutionary model of how some features of this complex system could have evolved.The mammalian RTL1 sequence [RTL1 sequences from the reference genomes of representative primates , rodents , afrotherians  and xenarthrans . Figure RTL1 genes Our analysis will distinguish between ‘autotargets’ and ‘allotargets’. An autotarget is a sequence that is directly complementary to a microRNA because it is transcribed from the opposite strand of the DNA double helix. Allotargets are sequences that are complementary to the microRNA’s seed but are not autotargets. The distinction between autotargets and allotargets is evolutionarily significant because changes to seed sequences are necessarily associated with corresponding changes to autotargets because microRNA and autotarget are transcribed from opposite strands of the Three of the antisense microRNAs have predicted allotargets on the sense mRNA Fig. : miR-136RTL1 locus. We acknowledge the limitations of non-experimental analyses but hope that they can provide some guidance for experiment. Experimental data on the interaction between mRNA and microRNAs at the RTL1 locus are currently limited. The microRNAs are known to initiate cutting of RTL1 mRNA but the specific sequences targeted by each microRNA are unknown. We identified potential targets by the criterion of perfect matches to seed sequences, but complementarity is not proof that an interaction occurs in vivo and may miss targets that lack perfect complementarity to the seed sequence . Is the cleavage of RTL1 mRNA by a microRNA mediated by its binding to its autotarget that is complementary to the entire length of the microRNA or by its binding to allotargets that are complementary only to the seed sequence?Our in silico predictions of intralocus allotargets hint at hidden complexities of RNA–RNA interactions (and possibly RNA–DNA interactions) at the RTL1 sequence [A notable feature of prior analyses of the sequence , 18 was RTL1 mRNA. This 33-nucleotide periodicity corresponds to one TRB unit. miR-432 is processed from within the complementary TRB array of antisense transcripts. We detected seven degenerate copies of TRB in the RTL1 genes of afrotherians, xenarthrans, humans and non-muroid rodents  to the miR-434-3p seed, three of them allotargets, and three perfect matches (AGUCGAG) to the miR-434-5p seed, two of them allotargets . As a consequence, the mouse Rtl1 mRNA (sense) contains seven perfect matches (CCAUCCA) to the seed of miR-432-3p and two perfect matches (CUCCAAG) to the seed of miR-432-3p. However, the TRB2 repeats disrupt formation of the pre-miR-432 hairpin. The rat Rtl1 gene also cannot produce pre-miR-432 but, in this case, because of deletions within the ancestral pre-miR-432 sequence. Deep-sequencing reported in miRBase detects miR-432 in neither rats nor mice [A 66-nucleotide sequence within the ancestral pre-miR-432 sequence has undergone six-fold tandem amplification in the nor mice . We haveTRB and TRC repeats exhibit interesting parallels  that is remarkable for a stretch of 442 nucleotides without a cytosine . At the amino acid level, this region encodes 115 glutamic acids, 14 aspartic acids, and 14 glycines. TRD repeats are absent from the Rtl1 genes of rats and non-rodents.Finally, the mouse RTL1 mRNA. An intriguing question is whether the principal selective forces in the fixation of these repeats acted on properties of the RNA sequence or properties of the encoded amino acids. Many proteins contain internal repeats with a variety of functions [The lengths of the various repeat-units are all multiples of three—TRA (12), TRB (33), TRB2 (66), TRC (24), TRD (33)—as required to preserve the open reading frame of unctions  but we cRtl1 are not associated with a microRNA. It is possible that these repeats evolved because of their contribution to the amino acid sequence of Rtl1 protein. Another possibility is that the repeats are associated with formation of an R-loop between the C-rich template DNA and G-rich RNA transcript. In this configuration, the G-rich non-template DNA might fold back upon itself to form G-quartet structures [Rtl1 mRNA.The TRD repeats of mouse ructures . Such antrans-acting and cis-acting factors in parental germlines: an imprinting control region (ICR) is differentially marked in male and female germlines by trans-acting factors, then the epigenetically-inherited state of the ICR determines expression of imprinted genes in cis in the next generation [trans-acting and cis-acting factors is crucial for understanding the evolution of genomic imprinting in mammals. If there is heterozygosity at a trans-acting locus that acts in the mother’s germline, an allele can influence gene expression in all of a mother’s offspring, not just the 50% that receive the trans-acting allele. By contrast, if there is heterozygosity at a cis-acting locus, then the effect is experienced only by those offspring that inherit the cis-acting allele. Therefore, a gene that acts in trans to imprint an ICR is subject to different selective forces than the ICR that acts in cis to determine gene expression in the next generation. This is a source of evolutionary conflict between maternal genes (in mothers) and maternally-derived genes (in offspring) [The establishment of imprints in mammalian genomes involves interactions between neration . The disfspring)  and has fspring) .trans-acting loci modify cis-acting loci in the haploid phase because trans-acting and cis-acting alleles are necessarily inherited together in products of syngamy. The establishment of epigenetic marks in gametophytes (haploid phase of plants) rather than sporophytes (diploid phase of plants) may explain why ‘imprinting’ genes are imprinted in plants, but not mammals, and why clusters of imprinted genes under cis control of an ICR have not been described in plants [trans-acting and cis-acting loci whose effects are restricted to the diploid generation in which the loci act.Conflict between imprinting genes and imprinted genes occurs because of their independent segregation at meiosis. Conflict is not predicted when n plants . Confliccum effects [trans-homolog interactions [RTL1 locus, do not map neatly onto the cis versus trans distinction: they occur at the same locus but not on the same chromosome. In this section, we present a model in which self-targeting microRNAs are derived from tandem repeats. The model illustrates how cum effects are subject to distinctive selective forces from cis and trans effects. The model was inspired by the observation that miR-432 and miR-434 are derived from tandemly-repeated sequences in the RTL1 gene.Genetic effects in which one allele influences the expression of its homolog have been labelled  effects  or transractions . Such efA) of RTL1 that contains a single copy of a partially palindromic sequence. In AA homozygotes, the maternal allele is expressed as an antisense non-coding RNA and the paternal allele is expressed as a sense RTL1 mRNA  in A*A** heterozygotes. Thus, maternal A** would enjoy the benefit of ‘altruistic’ restraint by paternal A*, but paternal A** would act ‘selfishly’ and not reciprocate (or incompletely reciprocate) when the roles were reversed.Green-beard effects are generally considered evolutionary unstable because of the threat of ‘false beards’: variants that enjoy the benefits of the altruism of other alleles but do not reciprocate when the roles are reversed. In terms of our model, a population fixed for A** would be an allele with a variant sense promoter that increased transcription of the paternally-expressed mRNA. This would favor an allele (A***) with a variant antisense promoter that increased transcription of the maternally-expressed microRNA. Such considerations predict an escalation in the strength of antagonistic promoters. Another possibility would be the acquisition, by the mRNA, of ‘decoy’ target sites for the microRNA that ‘sponge’ the microRNA but are resistant to cleavage.If one assumes that increased transcription increases the number of mRNAs that escape degradation, then the simplest example of RTL1 sequence because the model is iterable. One could think of this process as successive introductions of new traits for self-recognition: a green beard, followed by a blue mustache, followed by red whiskers. Once a trait is introduced, it should tend to be maintained by natural selection because changes to the target site on paternally-expressed mRNA to evade repression would be associated with changes to the maternally-expressed microRNAs which would thereby be less effective inhibitors of established alleles. This is a simple consequence of the complementarity of targeting and targeted sequences transcribed from opposite strands of the same double helix.The model presented above for the introduction of a single microRNA could potentially explain the origin of multiple self-targeting microRNAs encoded by the RTL1 locus. We conjecture that coordination between RTL1 alleles allows greater reproductive efficiency relative to control by a simple paternally-expressed demand enhancer. The cooperation posited here has some resemblances to the ‘coadaptation’ theory of genomic imprinting that posits an advantage from ‘matching’ of expressed alleles between parents and offspring [Maternal resources are predicted to be least efficiently distributed when allocation is controlled by paternally-expressed genes in offspring, more efficiently distributed when controlled by maternally-expressed genes in offspring, and most efficiently distributed when controlled by genes expressed in mothers . The levffspring , 48, altWe acknowledge the limitations of this model. An ideal population-genetic model would describe the dynamics of the system for all allele frequencies. We have found such models to be analytically intractable. It is difficult to model fitness trade-offs within families mediated by offspring demand in a population-genetic framework. Most previous models have avoided these complications by seeking an evolutionary stable strategy (ESS) that cannot be invaded by alternative strategies when an allele is near fixation in the population. We did not use an ESS approach because greenbeard effects are not predicted to be evolutionarily stable.RTL1 genomic sequence are, for the most part, highly conserved: pre-miR-136, pre-miR-127, and pre-miR-433 are highly similar in all eutherian RTL1 genes, as is pre-miR-432 (except in muroid rodents) and pre-miR-433 (except in xenarthrans where the sequence has not been found).Greenbeard altruism has been predicted to be vulnerable to exploitation by mutations that maintain the trait, and thus receive the benefits, but do not respond in kind . As a consequence, evolutionary models of greenbeard effects often predict periods of cooperation interspersed with periods of exploitation, accompanied by turnover in the tags used in self-recognition . Despitecum interactions at the RTL1 locus. First, the greenbeard triad of trait, recognition and response are all instantiated by the same short sequence and its antisense complement. ‘False beards’ may be difficult to evolve because mutational changes to autotargets that allow the paternally-expressed mRNA to evade inhibition by unmutated maternally-expressed microRNAs would come at the cost of a failure of maternally-expressed microRNAs with the mutated seed to inhibit paternally-expressed mRNAs with the unmutated target. Second, escalation in the strength of sense and antisense promoters increases the cost of loss of inhibition. Third, the accumulation of trans allotargets creates a selective force that opposes loss of the microRNA. Such allotargets can be considered to have ‘eavesdropped’ on the conversation between RTL1 sense and antisense transcripts.A number of factors may have stabilized RTL1 antisense microRNAs in most eutherian mammals, we find evidence of two episodes of rapid evolutionary change in the RTL1 sequence. First, we infer an early epoch of rapid change before the divergence of the major clades of extant eutherian mammals. During this period, the RTL1 gene originated from a retroelement, became subject to imprinted expression and antisense transcription, and gave rise to at least four self-targeting microRNAs. We are unable to tell whether these changes comprised a single episode of rapid change or multiple episodes. The details are lost in the fog of evolutionary time.Despite evolutionary conservation of the Rtl1 genes of muroid rodents. The neighborhood of the ancestral pre-miR-432 has undergone an expansion of TRA and TRC repeats in an ancestor of rats and mice and of TRB2 repeats in the mouse lineage. Moreover, numbers of TRA and TRC repeats differ between mouse and rat Rtl1 genes suggesting changes in one or both lineages have continued after rats and mice diverged. miR-434 is present, and miR-432 absent, in both rats and mice. We hypothesize that miR-434 functionally replaced miR-432 in self-targeting of Rtl1 mRNA. This replacement involved the loss of the miR-432 seed sequence and gain of the miR-434 seed. Our analysis has not resolved whether the gain of miR-434 and loss of miR-432 were evolutionarily contemporaneous or whether there was an extended period in which both microRNAs were produced. Thus, we do not know at what stage in the process miR-432-related sequences were deleted in the rat lineage and TRB2 repeats amplified in the mouse lineage. Some of these questions might be resolved by comparative studies of Rtl1 genes among muroid rodents.Second, we infer a relatively recent episode of rapid change in the Rtl1 genes show change in the number of TRC repeats continued after the origin of miR-434. One consequence of the amplification of TRC repeats has been the generation of additional allotargets for miR-434-5p and miR-434-3p. An unanswered question is whether these extra targets increase or decrease degradation of Rtl1 mRNA. Does the possession of multiple binding sites increase the probability that an mRNA will be degraded, or, do the extra allotargets function as ‘sponges’ [Rtl1 mRNAs that escape degradation?The origins of miR-432 and miR-434 were both associated with tandem duplications of short repeats and, in both cases, more repeats are present than the number required to generate the pre-miRNA hairpin. Furthermore, differences in the number of repeats between mouse and rat sponges’  that incRTL1 locus that are associated with tandem repeats. miR-127, miR-136, miR-431 and miR-433 are not associated with obvious repeats. Future work could explore whether these microRNAs also originated from tandem repeats, now highly degenerate, or originated in some other way.Our model of the origin of self-targeting miRNAs from tandem repeats applies directly to the origins of miR-432 and miR-434, the two microRNAs at the Studies of molecular evolution have focused, for the most part, on the coevolution of DNA and proteins. For example, whether a DNA sequence is under selection has been inferred from the ratio of synonymous to nonsynonymous changes in protein-coding sequences. Much of cellular biology has similarly focused on DNA and proteins. Under the traditional model of cellular control, DNA is transcribed as messenger RNAs that are translated as proteins which are the effective actors within cells. Some of these proteins function as transcription factors that bind to DNA promoters and determine where and when genes are expressed as protein-coding mRNAs.Noncoding RNAs are increasingly recognized as performing important roles in cellular control. Imprinted chromosomal regions, in particular, are home to many noncoding RNAs, including long noncoding RNAs (lncRNAs), microRNAs (miRNAs), and small nucleolar RNAs (snoRNAs) . The oneCum effects have been reported at multiple imprinted loci in addition to RTL1 [RTL1 locus. DLK1, the neighboring protein coding locus to RTL1, is a paternally-expressed gene (PEG) that is targeted in mice by multiple maternally-expressed microRNAs from the callipyge region [cum. Other examples of antisense miRNAs that target sense mRNAs in cum have been found in human and mouse genomes [cum effects is in its infancy.The sequence-specificity of interactions between non-coding RNAs and their nucleic acid targets promises a strategic richness of ‘clever tricks’ at imprinted loci. These include opportunities for ‘self-recognition’ in which an RNA recognizes the DNA sequence from which it was transcribed or recognizes RNAs transcribed from the other strand of the DNA double helix.  to RTL1 , 52–55 be region , another genomes  but noneRtl1 locus. The authors provide a convincing scenario for the establishment of the greenbeard effect for this locus. The manuscript is clearly written and the logic is easy to follow; I do not find specific flaws in the manuscript. I think this work is a substantial contribution towards evolutionary understanding of trans-homolog interactions (cum effect) at imprinted loci.Mainieri and Haig propose an evolutionary model for the emergence of self-targeting microRNAs from tandem repeats based on the analysis of the imprinted Rtl1 locus is to target the Rtl1 mRNA, thus miRNAs acting as a trait in the greenbeard effect and regulation of the target mRNA as a response. The fact that many miRNAs in this locus are conserved suggests that they have other (probably more prominent) functions. Could it be that the greenbeard effect at this locus has evolved further and inverted, i.e. that the primary function of Rtl1 mRNA is currently to regulate the activity of its miRNAs via a sponge effect? More discussion on this might be interesting.Figure 7 now Fig.  would beAuthors’ responseRtl1 mRNA acting as a sponge for the antisense miRNAs is an interesting one. We raised this possibility briefly in our discussion of the amplifications of TRC repeats in rodents. A related question is the conservation of allotargets in the Rtl1 mRNA for some of the antisense microRNAs. Are these allotargets conserved because they increase the number of target sites for cleaving the mRNA, because the allotargets ‘sponge’ the microRNAs, or for some more complex process of control (‘information processing’)? Consider the conserved allotarget of miR-136-3p that overlaps the Dicer cleavage site of pre-miR-127 , and it should also be important for other loci, as well as, potentially, human diseases and disorders that are mediated by alterations to genomic imprinting effects. The authors very nicely integrate the theory with the molecular data, and provide a useful set of relevant figures.callipyge system, in its relation to RTL1, for sheep (and humans), provided that they are of sufficient relevance. Second, there are rather too many Figures in the main text. Some of the less important of them  could be relegated to Supplementary material. The authors do need, however, to add a new figure that shows, as clearly and intuitively as possible, how their hypothesis of conflict works (how it evolved and how it is maintained). The writing in the Results is very dense and highly technical and detailed, and will be a real challenge for most readers - a figure will help considerably to make it penetrable. The authors might also add a Figure that depicted the green beard  effects, which will otherwise be hard for readers to conceptualize. Third, the Conclusions and the Results are not as clearly separable as they could be; conclusions can be quite short and describe the main points and implications, and what should be done next to evaluate the hypothesis further. Fourth, the authors should at least briefly discuss the roles of RTL1 in human disease, including infertility and cancer, and discuss how their proposed hypothesis may relate to disease susceptibilities and forms.The manuscript can be improved and made more accessible and useful in several ways. First, the authors need to provide more background regarding mechanisms of molecular conflict in imprinted-gene systems, and what the main unresolved questions are in this area of research. This should be done in the Abstract, and in the Introduction. Most readers will not be very familiar with imprinted genes, so the authors need to provide more background in the different ways that imprinted genes act in conflict (or cooperation) with one another, and how these known ways relate to the ways described here (which are rather different). The authors should also discuss polar overdominance and the Authors’ response.callipyge phenotype of sheep, added references to antagonistic effects of MEGs and PEGs in fetal growth in mice and humans, and added references to the effects of RTL1 and associated microRNAs in infertility and cancer. We have moved four Figures to the supplemental materials. The Results section is indeed dense and technical (it was not an easy section to write). We did our best to write simply about an intrinsically complex system. Figure We thank Professor Crespi for his kind words. In the Introduction, we have added discussion of polar overdominance and the Additional file 1: Figure S1. Conservation of sequences of (a) miR-136 (b) miR-127 (c) miR-433 (d) miR-432 and (e) mir-431. microRNAs (antisense) shown above mRNA sequences (sense); dots represent nucleotide identical to human RTL1. Homo , Ovies , Orycteropus , Dasypus . Figure S2. Self-complementarity of (a) miR-127-3p and (b) miR-433-3p. Predicted hairpin structures of pre-miR-127 and pre-miR-433 were obtained from miRDB [Figure S3. Alignment of seven 33-nucleotide TRB repeats for RTL1 genes (sense strand) of diverse eutherian species. Nucleotides in red and blue are complementary to pre-miR-432. Blue sequences are complementary to the seeds of miR-432-5p and miR-432-3p . The TRB repeats are disrupted in mouse and rat Rtl1 genes  The pre-miR-432 hairpin: CUUGGAG is the seed of miR-432-5p (appears twice); UGGAUGG is the seed of miR-432-3p; 33-nucleotide periodicity of TRB repeats indicated by successive arrow heads. (b) The pre-miR-434 hairpin: CUCGACU (appears twice) is the seed of miR-434-5p; UUGAACC (appears thrice) is the seed of miR-434-3p; 24-nucleotide periodicity of TRC repeats is indicated by successive arrow heads. The mature microRNAs processed from the hairpins are indicated by yellow. Structures of pre-miRNA hairpins from miRdb [om miRDB . The matom miRdb . (PDF 30


Viral vector-mediated gene therapy has grown by leaps and bounds over the past several years. Although the reasons for this progress are varied, a deeper understanding of the basic biology of the viruses, the identification of new and improved versions of viral vectors, and simply the vast experience gained by extensive testing in both animal models of disease and in clinical trials, have been key factors. Several studies have investigated the efficacy of adeno-associated viral (AAV) vectors in the mouse model of fragile X syndrome where AAVs have been used to express fragile X mental retardation protein (FMRP), which is missing or highly reduced in the disorder. These studies have demonstrated a range of efficacies in different tests from full correction, to partial rescue, to no effect. Here we provide a backdrop of recent advances in AAV gene therapy as applied to central nervous system disorders, outline the salient features of the fragile X studies, and discuss several key issues for moving forward. Collectively, the findings to date from the mouse studies on fragile X syndrome, and data from clinical trials testing AAVs in other neurological conditions, indicate that AAV-mediated gene therapy could be a viable strategy for treating fragile X syndrome. FMR1 premutation; approximately 40–75% of males and 16–20% of females with a premutation develop FXTAS [Fragile X syndrome (FXS) is a genetic disorder caused by a pathological expansion of a triplet repeat in the 5’ untranslated region of the FMR1 gene. Expansion from the normal 5–55 repeats to 200 or more causes hypermethylation of the gene promoter and shutdown of transcription and translation of the encoded protein, fragile X mental retardation protein (FMRP) . Althougop FXTAS .FMR1 gene undergoes alternative splicing to generate at least 16 mRNA isoforms [FMRP is a master regulator of gene expression in various organs including the brain, testes, and ovaries where it is highly expressed. The isoforms . FMRP isisoforms ,11, and isoforms . FMRP coisoforms ,14. Howeisoforms .Fmr1 KO mice, that FMRP plays an essential role in cerebellar inhibitory interneurons by both assisting in the trafficking of the channel to nerve terminals, and by facilitating Kv1.2 channel activity. In cerebellar basket cell interneurons, Kv1.2 controls (inhibits) gamma-aminobutyric acid (GABA) release from basket cell terminals [In addition to its mRNA binding role, FMRP also associates with and regulates other proteins directly. Salient examples include voltage-gated potassium channels which modulate the sodium-dependent action potential in neurons ,17,18. IThe clinical profile of FXS overlaps with that of autism spectrum disorders (ASD). Shared characteristics include impaired communication, sensory hypersensitivity, anxiety, stereotyped or repetitive behaviors, aggression, and cognitive impairment . HoweverB receptor. Progression to Phase 2 (mGluR5 antagonists) and Phase 3 (a GABAB agonist) clinical testing was based on extensive encouraging results from both in vitro tests, and in vivo animal analyses using the fragile X knockout mouse model. However, none of the clinical trials led to new drug approvals due in large part to lack of efficacy [Current pharmacotherapy for FXS still consists exclusively of symptomatic drug treatment. Examples include stimulants, antidepressants, antipsychotics, and valproate that are each somewhat effective in suppressing a subset of symptoms . Over thefficacy . A priori, a major motivation for pursuing small molecule drugs has been based on the idea that a particular receptor, enzyme, or other type of protein that is over- or under-expressed, or is over- or under-active, induces a major symptom or cluster of symptoms, and that when normalized, will result in major therapeutic improvements. The fundamental issue here stems from the difficulty in determining which of the many potential targets of FMRP, when corrected, will actually result in robust, measurable improvements in physiology, behavior, and health of persons with FXS.The failure of small molecule drugs in clinical trials to date might reflect, in part, the pleiotropic nature of FMRP. In light of the many varied roles of FMRP it should not be completely surprising that drugs that specifically block or activate individual receptors, enzymes, or other proteins may not be sufficient to provide a deeper and more comprehensive correction of the disorder. An alternative approach is to try to correct the basic underlying biochemical defect of the disorder-the absent or dramatically reduced levels of FMRP in the brain. Viral vector-mediated gene therapy is one potential avenue for rectifying the fundamental molecular defect in FXS. This approach may also be amenable to other genetic disorders associated with ASDs . The essRecombinant adeno-associated viral (AAV) vectors are currently the most widely employed class of viral vectors for gene therapy. Less commonly used vectors include adenovirus vectors and lentivirus-based vectors. The lentiviral vectors have been used successfully in ex vivo treatments, for example in treating X-linked cerebral adrenoleukodystrophy where therapy requires infusion of purified autologous CD34+ cells transduced with a lentiviral vector . NeverthLimitations of AAV vectors are the restricted room for target DNA insertion into the AAV vector and the presence of pre-existing circulating anti-AAV capsid antibodies in up to about half of the human population ,28. PreeFMR1 gene sequences, ssAAV vectors are deployable for all isoforms including the full-length isoform 1 [In addition to multiple serotypes, recombinant AAV vectors are of two main types; single-stranded vectors (ssAAV) and self-complementary vectors (scAAV). The former possesses a larger insertion capacity of about 4.6 kilobases of DNA, while the latter has a very limited capacity restricted to a maximum of 2.4 kilobases, but is more efficient at expressing the inserted transgene . It shousoform 1 ,30. The soform 1 . So far,soform 1 .Recombinant AAV vectors used for gene therapy are packaged into virus particles and subjected to purification by density gradient centrifugation  followed by high performance liquid chromatography with ion exchange chromatography. After binding to an AAV receptor protein on the surface of cells, the viral particle enters the cell and begins to transcribe and translate copies of the encoded transgene, but the virus itself does not replicate. Each AAV serotype has one or more receptor protein(s) that is primarily responsible for mediating viral uptake into cells and tissues . The choIn addition to relatively low tissue and cell-type selectivity imparted by most of the natural viral serotypes, further restriction of transgene expression is achieved via the use of customized gene promoters. Several CNS specific or selective promoters have been tested in AAV gene therapy experiments in preclinical animal studies. Examples include the synapsin promoter for neuron-selective expression , the dlxAnother very attractive aspect of AAV vectors for treating CNS disorders is the capacity for long-term expression of the therapeutic protein. Upon administration, recombinant AAV-mediated therapeutic protein expression gradually ramps up over the first few weeks after injection and plateaus about 3–4 weeks post-injection. The epichromosomal presence of the AAV is static and can translate into long-term expression of the desired transgene. In tissues containing dividing cells with cellular turnover, expression levels will dwindle over time. In non-dividing long-living cells like differentiated neurons, recombinant transgenes are typically expressed for years. For example, transgenes expressed from injected AAVs have been shown to persist in the primate brain and maintain a therapeutic effect for up to 10 years ,37,38. TAs noted above, developing AAV-FMRP gene therapy for FXS presents a numbers of issues that need to be resolved for moving this potential biological therapeutic drug from the laboratory to clinical trials and beyond. At the top of the list are issues associated with CNS delivery of biologically-based therapeutic drugs such as viral vectors. Because of the pan distribution of FMRP throughout the brain, direct injections into the parenchyma are largely precluded due to lack of sufficient spread of the virus to other brain regions. The logical solution would be to administer the vector systemically, for example, via intravenous injection. Several obvious problems here include the very large quantities of a very expensive vector that need to be injected, much or most of which binds to and is taken up by peripheral organs such as the liver and heart that act as sinks for several types of viral vectors, and the potential complications of virus-induced side effects from treating patients with the large doses that would be required to obtain adequate FMRP expression levels in the CNS . A potential solution is to infuse the vector into the cerebral spinal fluid (CSF) through the spinal canal , by intra-cisterna magna (i.c.m.) injection near the base of the skull, or by intracerebral ventricular injection (i.c.v.). All three of these routes are applicable to studies in experimental mammals, while i.t. injections have been most the widely used mode for administering drugs into the CSF in humans and non-human primates ,41,42,43A second issue that must be dealt with is proper dosing with the goal of achieving normal “wild-type” levels of FMRP in as much of the brain as possible. Achieving vector-derived FMRP expression at an adequate level in the brain, and in the appropriate types of cells are important parameters that are discussed further below. Viral vectors differ from small molecule drugs in that they are typically given only once or a few times, and are not easily amenable to dose modulation. This means that the injected dose(s) must be carefully established prior to initiation of treatment because with current vector systems, there is no opportunity for reducing the dosage and level of transgene expression downward. Additional dosing to achieve a higher level of expression can be accomplished by one or more subsequent treatments, but as noted above this comes with increased risk of inducing the immune system to generate anti-capsid antibodies that can neutralize or hinder the efficacy of the viral vector therapeutic effects  [Fmr1 coding sequence, and a FLAG epitope tag. The virus was injected bilaterally directly into the hippocampus of 5 week-old Fmr1 KO mice. At the cellular level, Fmr1 KO mice have been shown to display abnormal synaptic plasticity as manifested by impaired long-term potentiation and enhanced long-term depression. Zeier et al. (2009) conducted brain slice recordings of extracellular postsynaptic field potentials from area CA1 of the hippocampus 3–5 weeks post-injection [Fmr1 KO mice treated with AAV5-FMRP showed correction of abnormally enhanced long-term depression. This finding is important because altered synaptic plasticity in FXS is thought to contribute to the cognitive deficits in the disorder.To date, studies using viral vector gene therapy in the . (2009) . This grnjection . Fmr1 KOFmr1 isoform 1 under the control of the neuron-specific synapsin promoter. Anatomical and cellular analyses using immunocytochemistry revealed transgene expression in neurons in various forebrain regions with lower expression in more caudal areas of the CNS. The observed rostral-to-caudal gradient was expected based of the location of the injection site in the lateral ventricles. Quantitative western blotting using an anti-FMRP antibody of samples collected at Postnatal Days 30 to 60 showed a range of transgene expression levels across different brain regions, from a high of 71% in the cerebral cortex to a low of 18% in the striatum compared to baseline wild-type expression. Low but detectable levels were also seen in the cerebellum. AAV-FMRP expression was still detected at 7-months post-injection demonstrating the stability of the FMRP transgene.The next published study  was conducted by our group which utilized an AAV9 vector administered by i.c.v. injection at Postnatal Day 5 . Here, aFmr1 KO mice, motor activity is increased relative to wild-type mice, marble burying reflecting repetitive behavior, is increased, social dominance as determined by the tube test is decreased, as is the number of ultrasonic vocalizations. Treatment of KO mice with AAV-FMRP normalized stereotyped behavior and social dominance, whereas no significant changes were observed in motor activity or the frequency of vocalizations. Fmr1 KOs but not wild-type mice are highly susceptible to sound-induced (audiogenic) seizures. This is a dramatic and robust endophenotype of Fmr1 mice in which typically 50–75% of the tested KO mice undergo seizures [For behavioral analyses, a range of tasks were employed to judge normalization after AAV-FMRP . In uninseizures ,54. GholFmr1 KO mouse neonates and obtained FMRP expression mainly in forebrain regions; this translated into correction of repetitive behavior and social dominance but not motor hyperactivity, abnormal calling frequency, or increased seizure susceptibility [To summarize, the Gholizadeh et al. (2014) study injected AAV9-FMRP with a neuron-specific promoter into the CSF of tibility .The third published study to date was also carried out by our group. In the Arsenault et al. (2016) study we again used AAV9 and mouse isoform 1 driven by neuron-specific promoters, but instead of injecting control mice with phosphate-buffered saline, an AAV9 carrying no transgene was used as the negative control  . This stFmr1 KO mice display an increase in the number entries and the time spent in the open arms of the maze [Fmr1 KO mice, and perhaps other lines of mice with autistic features, may instead reflect cognitive impairment [A second objective was to extend behavioral testing to additional tasks not carried out in the previous Gholizadeh et al. (2014) study . The newthe maze ,56,57. Tthe maze ,59. Tradpairment . In thispairment ,61). Fmr1 KO mice. We also ascertained that the transcription modulator MeCP2, the mutation of which causes Rett Syndrome and is associated with some autistic features, is a substrate for FMRP . We found that MeCP2 protein levels are elevated in the Fmr1 KO mouse brain. Both PSD-95 and MeCP2 proteins reverted to wild-type levels 4 weeks after injection with AAV-FMRP under the control of the synapsin promoter. Therefore, at the biochemical level, treatment with AAV-FMRP was capable of correcting the expression of key proteins that are regulated by FMRP.A third objective of the Arsenault et al. (2016) study was to examine the normalization of FMRP substrates in the CNS after treatment . The higFmr1 KO mice. We compared five treatment groups injected with one of three test vectors: wild-type (WT) mice treated with AAV-EV (the control baseline group), WT mice treated with the AAV-FMRP vector, and Fmr1 mice treated with AAV-EV (control baseline group of impaired KO mice), AAV-FMRP (the therapeutic treatment group), or the AAV-FMRP high-expressing vector representing a high over-expression group. This allowed for an assessment of the effects of a wide range of FMRP levels in the brain. Distillation of the results indicated that partial rescue of the Fmr1 phenotype was observed in mice with forebrain FMRP levels of approximately 35–70% of wild-type, while moderate over-expression of up to approximately 120–140% did not induce behavioral abnormalities. However, massive over-expression of 200–600% of normal wild-type levels induced aberrant behaviors such as hyperactivity and an abnormally reduced startle response. The take-home message here is that modest under-expression appears to produce some therapeutic effects, while modest over-expression of FMRP does not appear to induce pathology. These findings suggest that a fairly wide range of CNS FMRP levels could translate into therapeutic benefits. Finally, the fourth major objective was to probe the consequences of under and over-expression of FMRP relative to normal wild-type levels in the CNS. The purpose of this part of the study was to conduct the first dose ranging study of FMRP in The three studies reviewed above that explored the efficacy of AAV-FMRP therapy in the mouse KO model have collectively demonstrated a general proof-of-principle whereby AAV-FMRP fully or partially corrected abnormalities on several behavioral tasks and in vitro assays conducted at the cellular and biochemical levels. However, considering the high bar likely to be set by regulatory agencies for moving forward with a Phase 1 clinical trial for FXS, additional evidence supporting the efficacy and safety of viral vector-mediated gene therapy would bolster the case for proceeding to a clinical trial. To date, clinical trials for CNS disorders using viral vector-mediated gene transfer were approved by regulatory agencies, in part, because they addressed unmet needs for treating severe degenerative disorders such as Parkinson’s and Alzheimer’s disease, or neurodevelopmental disorders that cause paralysis and/or death such as spinal muscular atrophy type 1 and giant axon neuropathy. In contrast, FXS and most other forms of autism spectrum disorders do not lead to dementia, severe paralysis, or premature death. Nevertheless, FXS and ASDs are life-long disorders in which the majority of affected individuals display moderate to severe cognitive impairment along with a slew of additional medical problems. Most will need some type of life-long care from an early age. Therefore, the discovery and development of an effective treatment would have enormous benefits not only for those with FXS, but also their families and health care funders. Optimization of several technical parameters should also be considered in moving forward towards clinical testing. First, further assessment of drug delivery methods could be beneficial. Ultimately, experimental treatment methods in the laboratory should be representative of routes of administration that will be used in patients. Assessing additional drug delivery protocols that could provide more wide-spread diffusion of the AAV vectors after injection into the CSF, especially to more caudal regions of the brain, would likely boost the therapeutic response. Simply administering a larger amount of the virus from a single point-source injection may not be a solution because of the liability of transgene over-expression near the needle injection site. However, injecting at more than one site might provide a work-around. Our preliminary observations in mouse brain suggest that simultaneously injecting the vector into the CSF at more than one site, for example, an i.c.v. injection together with an i.c.m. (or i.t.) injection, may enhance transgene coverage in the brain , rats are generally thought to be smarter and capable of performing more difficult cognitive tasks [Third, in addition to ongoing work in mice, testing AAV-FMRP distribution and efficacy in additional species could provide further confidence in establishing AAV-FMRP as a viable therapeutic agent. In terms of an alternative disease model, the logical choice would be the ve tasks . Deficieve tasks ,73,74. Fmr1 KO rat is relatively uncharacterized compared to the Fmr1 KO mouse. Nevertheless, the Fmr1 KO rat has been reported to display impairments or abnormalities on tests measuring sensory perception, communication, cognition, and other functions [Fmr1 KO rats, despite normal speech discrimination behavior. Evoked potentials and spiking activity in response to speech sounds, noise burst trains, and tones were degraded in primary auditory cortex and the anterior and ventral auditory fields compared to wild-type rats [Because it is much newer on the scene, the unctions ,79,80,81unctions . Juvenilype rats .Fmr1 knockout rats [Fmr1 KO rats showed that during quiet rest when activity in wild-type rats became dominated by the inactivated state (3–9 Hz), cortical activity in the KO rats remained activated, resulting in increased high-frequency and reduced low-frequency power during rest [Fmr1 KO rats, particularly between fast-spiking inhibitory interneurons. The findings from this study, together with data from EEG analyses conducted in Fmr1 KO mice [Fmr1 KO rat is likely to be a useful addition for testing AAV-FMRP efficacy, particularly in probing cognitive and sensory functions.Abnormalities were also documented in an analysis of neuronal morphology and neurochemistry in the auditory brainstem of out rats , and impout rats . Robust out rats . Finallying rest . Moreove KO mice , and fro KO mice , are impFmr1 KO mouse. Whether or not this is an issue for the treatment of human subjects remains unknown. Injecting AAV-FMRP via the intra-cisterna magna route with (Gene therapy studies conducted so far have used the mouse model of FXS and demonstrated full or partial correction of selected deficits after early postnatal treatment with AAV-FMRP. Two of the published studies injected AAV-FMRP vectors containing neuron-specific promoters into the CSF of the neonatal mouse brain to drive FMRP transgene expression in neurons ,30. The ute with  or withoFmr1 conditional knockout and conditional restoration mice have demonstrated that the selective deletion of FMRP in glia or in the prefrontal cortex after birth results in cellular and behavioral deficits [Moving forward, useful experimentation should encompass exploring the therapeutic effects of administering AAV-FMRP at later time points after birth to more closely mimic a clinical trial situation. Additionally, results from the study of deficits ,84. The Additionally, more in-depth characterization of rodent tests that are translatable to a clinical trial, such as prepulse inhibition and EEG, and the effects of AAV-FMRP on normalization of these parameters, could prove essential in eventual approval of this experimental biological therapeutic drug. Finally, encouraging positive results from recent clinical trials using AAVs in severe childhood diseases have generated extremely useful information from several technical standpoints ranging from drug delivery and drug dosage to post-treatment care and follow-up. Overall, the momentum attained in the field of viral vector-mediated gene therapy has now created further incentive to continue to develop this technology for treating not only FXS, but also other neurodevelopmental disorders caused by known gene mutations.


Despite effective factor replacement and various treatment schedules, there remain several challenges and unmet needs in the prophylactic treatment of hemophilia limiting its adoption and thereby posing an increased risk of spontaneous bleeding. In this regard, extended half-life (EHL) recombinant factor VIII (rFVIII) and factor IX (rFIX) products promise optimal prophylaxis by decreasing the dose frequency, increasing the compliance, and improving the quality of life without compromising safety and efficacy. EHL products might lead to higher trough levels without increasing infusion frequency, or could facilitate the ability to maintain trough levels while reducing infusion frequency. This paper aims to provide a comprehensive review of the rationale for developing EHL coagulation factors and their utility in the management of hemophilia, with special emphasis on optimal techniques for half-life extension and criteria for defining EHL coagulation factors, as well as indications, efficacy, and safety issues of the currently available EHL-rFVIII and EHL-rFIX products. Potential impacts of these factors on quality of life, health economics, and immune tolerance treatment will also be discussed alongside the challenges in pharmacokinetic-driven prophylaxis and difficulties in monitoring the EHL products with laboratory assays. Hemophilia A and B are X-linked monogenic inborn coagulation defects that lead to deficiencies of factor VIII (FVIII) and factor IX (FIX) in approximately 1 of 5000 and 1 of 30,000 male live births, respectively ,3.The disease phenotype is characterized by recurrent spontaneous or traumatic bleeding episodes predominantly involving the weight-bearing joints, skeletal muscle, and soft tissues. Intracranial and retroperitoneal hematomas are rare but life-threatening complications of severe hemophilia ,3. The bReplacement of the missing factor constitutes the mainstay of hemophilia treatment. Factor replacement is given either on demand to treat acute bleeding or prophylactically to prevent bleeding ,6. In seRegular prophylactic factor replacement to maintain circulating factor levels of >1 IU/dL (1%) has been recommended as the optimal therapy for people with severe hemophilia, based on evidence showing that prophylaxis is associated with substantial reduction in bleeding episodes and related complications and consequently with an improvement in the quality of life and life expectancy ,10,11.In the prophylactic setting, people with severe hemophilia A usually require intravenous injections three times a week, while those with severe hemophilia B are usually treated twice weekly, owing to the longer half-life of FIX compared to FVIII (18-20 h vs. 8-12 h) [Due to the relatively short half-life of conventional factor concentrates, frequent intravenous administrations are required to maintain plasma factor levels above the target threshold level to avoid bleeding, and this necessitates frequent injections ,14. The Much effort has been devoted to the optimization of the pharmacokinetics (PKs) of recombinant factors by molecular modifications to achieve extended half-life (EHL) FVIII and FIX products ,23,26,27During the past decade numerous techniques have been invented for the development of EHL-rFVIII and -rFIX molecules, all of which principally exert their effect by decreasing the clearance of the factors. A combination of reduced proteolysis in peripheral blood, decreased renal and hepatic elimination, and decreased receptor-mediated endocytosis usually results in prolongation of the half-life of the factor . SeveralThis paper aims to provide a comprehensive review of the rationale for developing EHL coagulation factors and their utility in the management of hemophilia, with special emphasis on optimal techniques for half-life extension and criteria for defining EHL coagulation factors, as well as indications, efficacy, and safety issues of the currently available EHL-rFVIII and EHL-rFIX products. Potential impacts of these factors on quality of life, health economics, and immune tolerance treatment will be also be discussed alongside the challenges in PK-driven prophylaxis and difficulties in monitoring EHL products with laboratory assays.Management of hemophilia mainly depends on replacing the missing coagulation factor to stop (episodic or on-demand therapy) or to prevent (prophylaxis) bleeding episodes ,5. The cFVIII and FIX are large, complex proteins with relatively short half-lives, necessitating frequent dosing to maintain therapeutic levels. EHL coagulation factors are designed to have prolonged half-lives through some structural modifications, such as chemical alterations or fusion of the factor protein to another molecule with a longer half-life. In theory, an extended half-life product is expected to result in better adherence to treatment and improved prophylactic outcomes by allowing for less frequent injections .The optimal method for half-life extension should not cause any change in the biological activity and safety of the coagulation factor . The defi) covalent attachment of the coagulation factor to polyethylene glycol  to reduce interaction with clearance receptors; ii) integrating the coagulation factor with the fragmented crystallizable (Fc) portion of the immunoglobulin G1 (IgG1) molecule to divert the molecule away from lysosomal degradation to delay its clearance or iii) combining the coagulation factor and recombinant albumin, to rescue endocytosed proteins from the intracellular degradation pathway; and iv) single-chain technology for augmenting the stability of the molecule [The strategies to prolong the half-life of recombinant coagulation factors include molecule ,19,51,52The pharmacokinetic and pharmacodynamic properties of the coagulation factors can be changed through PEGylation, which involves the covalent binding of PEG to FVIII or FIX . The cirPEGylated factors have been reported to have a half-life prolongation of about 1.5-fold when compared to the standard half-life (SHL) factors. Excellent safety and efficacy data in previously treated adults and children with severe hemophilia A have been reported with EHL factors. No inhibitory antibodies have been identified to FVIII, the PEGylated product, or to PEG ,54,55,56Fusion protein technology involves genetic fusion with a protein that has a particularly long half-life, such as immunoglobulins (Fc fusion) or albumin ,60,61. AAlbumin fusion technology has recently been utilized for the prolongation of the half-life of rFIX . The meaThe long-term efficacy and safety of rFVIII-Fc in the management of bleeding and prophylaxis of previously treated patients (PTPs) with severe hemophilia A have been confirmed by several studies, including pivotal phase III trials (A-LONG) done in adults and adolescents aged >12 years, the Kids A-LONG trial performed in children aged ≤12 years, and ASPIRE, a recently completed extension study ,34,63,64Human FVIII is a heterodimeric structure consisting of a heavy chain (A1-A2-B domains) and a light chain (A3-C1-C2 domains) attached to each other by noncovalent bonds, which makes it relatively unstable and easily dissociable to inactivated FVIII chains ,23,49. AThree EHL-rFIX products have completed phase 3 clinical studies and are licensed for adolescent and adult patients ,41, inclThere are four EHL-rFVIII products that have completed phase 3 clinical studies. rFVIIIFc   and octoConsidering the mechanisms of half-life extension in rFIX concentrates, rFIX-Fc (Alprolix) fuses the Fc immunoglobulin region with FIX, while rFIX-FP (Idelvion) combines FIX with albumin and N9-GP (Rebinyn/Refixia) is a PEGylated version of FIX. As with EHL-rFIX concentrates, EHL-rFVIII concentrates are also based on Fc fusion  or PEGylation . BAY 94-9027 and N8-GP are B-domain-deleted (BDD) rFVIII, whereas BAX 855 is a full-length rFVIII ,41,56,68The main characteristics of EHL concentrates are provided in The half-life extension of EHL-rFVIII products is in the range of 1.4- to 1.6-fold and the annualized bleeding rates (ABRs) were below 4, ranging from 1.3 to 3.6. Bleeds were treated successfully with EHL-rFVIII, resolving with one or two injections in more than 96% of episodes. Hemostatic efficacy was rated as good or excellent in more than 90% of the bleeding episodes. No inhibitor development has been reported in clinical trials with the EHL-FVIII products .Data on the use of EHL products in the pediatric age group ,69,70,71The safety and efficacy as well as the PK profile of rVIII-SingleChain have been studied within the framework of a clinical trial program called AFFINITY, consisting of a series of phase I/III studies ,66,72. PThe advent of EHL recombinant factors has been an important evolution in concentrate manufacturing, providing a new treatment tool for individualized hemophilia care ,74,75. HIdeally, EHL recombinant factors should allow reduced dosing frequency with retention of hemostatic efficacy compared to SHL recombinant factors for the majority of patients . HoweverGiven the tight non-covalent association of FVIII with VWF in the circulation, which imposes a biological limit on the extension of the half-life of FVIII beyond that of VWF, the EHL-rFVIII products have not had an equally substantial improvement in half-life as observed with EHL-rFIX products ,77. EHL-i) the product should be designed and engineered with relevant technology used to extend the circulating biological half-life; ii) the difference from the SHL-rFVIII comparator should be demonstrated for the majority of patients according to the proposed “bio-difference” criterion based on the lower limit of the 90% CI for the AUC ratio being above the FDA/EMA cut-off for bioequivalence (1.25 or 125%); and iii) a half-life ratio of 1.3 or higher, based on modeling, should be achieved [However, this is not always the case for FVIII products. In a modeling study designed to identify the minimum half-life extension ratio required for a reduction in dosing frequency while maintaining the proportion of patients with plasma rFVIII levels >1 IU/dL with no increase in the total weekly dose, the authors found that a meaningful reduction in the infusion burden of an EHL-rFVIII product (relative to a standard rFVIII) is only possible when the half-life extension ratio is ≥1.3 . In addiachieved ,79.®), based on the 90% CIs for the AUC ratio extending below 1.25 and a half-life extension ratio of 1.09 when compared to Advate®. This suggests that rFVIII-SingleChain may behave like standard rFVIII in some patients despite its modified PK characteristics [BAX 855 and rFVIIIFc have been reported to clearly comply with all 3 of these criteria, while rFVIII-SingleChain failed to fulfill the criteria since it cannot be fully differentiated from the standard rFVIII , “PK-driven” , “phenotype-driven” , and “convenience-driven”  strategies have been used for the prophylaxis regimens in pivotal clinical trial programs .There might be a concern regarding inhibitor development when switching between different FVIII concentrates as product type is one of treatment-related factors for inhibitor development . Recent Accordingly, data from the ALONG trial showed that 30% of hemophilia A patients in the individualized prophylaxis arm achieved 5-day dosing intervals in the last 3 months of the study . In the A 30% lower total weekly dose of rFVIIIFc has been shown to be likely to give the same FVIII exposure considering that rFVIIIFc has a 30% lower clearance when compared to rFVIII . Based oWhile the primary focus has been the potential use of EHL factor concentrates in patients with severe hemophilia, the utility of EHL products for the management of bleeding in patients with mild and moderate deficiencies has also been considered .Further support with real-life data on the potential advantages of EHL factor concentrates may enable identifying the needs and characteristics of individual patients and the difference in the behavior of SHL-FVIII products as compared to EHL products. This may help guide clinicians when switching hemophilia A patients from conventional FVIII to EHL products.Nonetheless, it should be noted that commenting on the comparative efficacy of new long-acting therapies is not possible due to the lack of head-to-head studies. Furthermore, it is difficult to compare different EHL products with the current clinical data since the published studies greatly vary with regard to study populations, study designs, and protocols and they evaluate outcomes using different end-points such as ABR, inhibitor development, number of breakthrough bleeds, or dosing intervals.Prophylaxis is considered as the gold standard for countries in which it is economically affordable ,85,86, aEHL factors offer better protection from bleeding while reducing the frequency of injections and allowing trough activity levels to remain above key thresholds for longer periods relative to conventional factor products . Thus, lIn two phase III studies done with rFVIIIFc and rFIXFc, HRQoL was assessed in adults with severe hemophilia A and B, respectively, who received prophylactic or episodic factor replacement regimens ,37. The In an analysis of the potential financial consequences of introducing rFVIIIFc to a private payer system in the United States, rFVIIIFc was anticipated to have a budget impact of 1.4% across 2 years for a private payer population of 1,000,000 through reducing the ABR by approximately 3.1 bleeds per individual patient with hemophilia A . The totFor prophylaxis, the cost per bleed avoided after the introduction of rFVIIIFc was estimated to be 1974$ in year 1 and 1808$ in year 2, while the small decrease in cost per bleed avoided over time was considered to be associated with the likelihood of patients uncontrolled on a fixed prophylaxis regimen to switch to an individualized regimen in year 2, resulting in a more efficient use of factor therapy .In the scenario without rFVIIIFc, the annual cost for the management of the estimated annual 388 bleeding episodes was 2,044,868$, which equates to each bleeding episode costing approximately 5270$. The cost per bleed avoided with rFVIIIFc on the market was approximately 1891$, indicating that prophylaxis with rFVIIIFc provides good value for money in the prevention of joint bleeds and related comorbidities .The introduction of rFVIIIFc to a private payer system is anticipated to have a minimal budget impact while reducing the ABR, alongside a potential for reduced dosing schedule required for rFVIIIFc and reduction in total factor consumption, facilitating adherence to prophylaxis regimens with a likely positive impact on patient quality of life and economic burden .Occurring in up to 30% of patients with severe hemophilia A, the development of alloantibodies (inhibitors) directed against FVIII is the most serious complication of replacement therapy , leadingThe current management of patients with inhibitor development involves treating acute bleeding with agents that bypass the need for FVIII or FIX, i.e. activated prothrombin complex concentrate or activated recombinant factor FVII, and using immune tolerance induction (ITI) in order to eradicate the inhibitory antibodies . SeveralWhile several meta-analyses failed to demonstrate a significant difference of inhibitor development in patients treated with recombinant FVIII compared to plasma-derived products, a randomized prospective study revealed that the use of recombinant products in previously untreated patients (PUPs) was associated with a 1.8 times greater risk of inhibitor development compared to plasma-derived products . There iA potential role for EHL factors for the induction of immune tolerance has also been suggested ,52. A seProphylactic dosing in severe hemophilia is generally tailored according to the individual needs of the patients. Tailoring of treatment has been guided by either clinical bleeding phenotype or individual PKs of a particular factor concentrate in a patient .PK-tailored prophylaxis was shown to have superior hemostatic efficacy compared to on-demand treatment, along with decreased factor consumption . PK-tailIndividualized PK-based dosing is considered as an alternative option for maintaining a predetermined factor trough level ,95. HoweIn addition, there are several challenges with PK-guided prophylaxis when EHL factor concentrates are used . In contGiven the unpredictable impact of bioengineered products on individual patients, increased knowledge on the PK parameters of new anti-hemophilic molecules with prolonged half-lives will improve tailored prophylaxis based on individual needs and PK characteristics, offering new possibilities for effective prophylaxis ,19. PopuMonitoring factor levels through laboratory assays is an important part of ensuring patient safety during hemophilia management. Commonly used laboratory assays for measuring FVIII or FIX activity may not be the optimal method for some EHL-rFVIII or -rFIX products, such as those modified through PEGylation or fusion to albumin or immunoglobulin ,101.While measurement of recombinant coagulation factor concentrates has always been complicated by the discordance between the measurements carried out with different types of assays , the molApart from the molecular modifications done to prolong the half-lives of the clotting factors, various properties of the original molecules themselves, as well as those of the assays, along with the use of different calibration methods contribute to the discrepancies in determining the plasma factor activity levels ,102.Chromogenic assays show less variability than one-stage assays in the measurement of FVIII activity levels, possibly due to a restricted choice of available assay kits and reagents ,104. ComHence, chromogenic assays are considered as the assays of choice for monitoring patients treated with several EHL-rFVIII or -rFIX concentrates. However, there are several challenges associated with the implementation of chromogenic assays in routine clinical laboratory practice, including increased expenses and technical complexity as well as the higher inter-laboratory variability at very low factor levels when compared to one-stage assays ,103,106.Concordance between assays used in laboratories and by pharmaceutical companies to measure the potency of a product, effective communication between the laboratories and the clinicians, and conveyance of relevant information by companies for correct monitoring of their products to both local laboratories and clinicians are essential for proper monitoring ,101. AddEHL factor concentrates have been shown to be well tolerated, safe, and efficacious in the treatment and prevention of bleeding episodes in people with hemophilia. These concentrates have the potential to induce higher trough levels with less frequent injections; thus, they reduce the infusion burden and facilitate adherence to prophylactic regimens ,76,85. MGiven the evolutions in the treatment of hemophilia in recent years with the advent of multiple non-replacement treatment options entering the market, clinicians and patients now face the prospect of having a variety of choices for individualizing treatments according to their needs . EHL-rFI


OBJECTIVES/SPECIFIC AIMS: Morphea  is an autoimmune disease characterized that is widely thought to have a monophasic course, in which an initial period of inflammation (activity) ultimately results in scarring, atrophy, and functional impairment (damage). Understanding the long-term clinical course of morphea is important for the planning of future interventional studies, and as a tool for clinicians in determining risk for poor disease outcomes. METHODS/STUDY POPULATION: We conducted a prospective cohort study of 130 participants enrolled in the Morphea in Children and Adults Cohort over a median follow-up time of 4.3 years, to determine the rates of response to treatment and disease recurrence as measured by the Localized Scleroderma Cutaneous Assessment Tool (LoSCAT). To determine risk factors for recurrence of disease activity, survival analysis using the log-rank test was used to compare subgroups by morphea type, therapy, and age at disease onset. RESULTS/ANTICIPATED RESULTS: Within a 1-year follow-up period, 66% of patients treated with methotrexate and 46% of patients with UVA1 phototherapy had achieved complete response to treatment. In patients who had achieved response to treatment, 29% experienced disease recurrence at an average of 1.7 years after documented disease inactivity. Patients with generalized morphea experienced higher recurrence rates than those with linear morphea , and those treated with UVA1 phototherapy had higher recurrence over those treated with methotrexate . In patients with follow-up periods longer than 5 years (n=50), disease recurrence was observed in 44% of patients and the majority of recurrence represented new activity in an area of pre-existing morphea (82%). DISCUSSION/SIGNIFICANCE OF IMPACT: This study highlights the previously under-studied dynamic long-term course of morphea, and identifies the clinical characteristics that predispose patients to having a relapsing-remitting course. We conclude that patients with morphea should be followed closely over time even in the absence of disease activity due to the potential for disease recurrence.


Nuclear magnetic resonance (NMR) spectroscopy is one of the most utilized and informative analytical techniques for investigating glycosaminoglycan (GAG)-protein complexes. NMR methods that are commonly applied to GAG-protein systems include chemical shift perturbation, saturation transfer difference, and transferred nuclear Overhauser effect. Although these NMR methods have revealed valuable insight into the protein-GAG complexes, elucidating high-resolution structural and dynamic information of these often transient interactions remains challenging. In addition, preparation of structurally homogeneous and isotopically enriched GAG ligands for structural investigations continues to be laborious. As a result, understanding of the structure-activity relationship of GAGs is still primitive. To overcome these deficiencies, several innovative NMR techniques have been developed lately. Here, we review some of the commonly used techniques along with more novel methods such as waterLOGSY and experiments to examine structure and dynamic of lysine and arginine side chains to identify GAG-binding sites. We will also present the latest technology that is used to produce isotopically enriched as well as paramagnetically tagged GAG ligands. Recent results that were obtained from solid-state NMR of amyloid’s interaction with GAG are also presented together with a brief discussion on computer assisted modeling of GAG-protein complexes using sparse experimental data. Glycosaminoglycans (GAGs) are linear, anionic, high molecular weight (MW) polysaccharides that are composed of repeating disaccharide building blocks of alternating hexosamine and uronic acid/galactose units . The monAmong all glycoconjugates in the glycocalix, GAGs are likely to be the most important player of the extracellular matrix in terms of protein interaction and regulation. This is because of two essential characteristics of these sulfated heteropolysaccharides: (i) they are ubiquitous to all eukaryotic cells; and, (ii) their highly complex structure gives rise to unique motifs that may have specific interactions with proteins. Hence, knowledge about the structural biology of GAG-protein interactions is extremely relevant. Three major goals exist in the current science of GAG-protein interactions: (i) identification of novel GAG sequences recognized specifically by proteins; (ii) identification of the potential GAG-binding site(s) in GAG-binding proteins; and, (iii) understand how GAG-induced changes in protein oligomerization and conformation regulate physiological activity.One analytical technique that is capable of studying the various aspects of GAG-protein interactions including the three above-mentioned goals is nuclear magnetic resonance (NMR), whose versatility has made it a popular choice among researchers interested in GAG-binding proteins. NMR-oriented studies have been designed to elucidate multiple aspects of GAG-protein interactions, not only through the individual protein and ligand point-of-views but also through the perspective of the resultant complexes in solution, including their conformational fluctuations from the ns-to-ms timescale.In this review, we want to offer an overview ofthe potentialsof this analytical technique for advancing studies of GAG-protein interactions. Although discussion on the most common NMR-based methods that are utilized to investigate GAG-protein interactions, such as chemical shift perturbation, saturation transferred difference, and transferred nuclear Overhauser effect, are presented, attention is also given to newer methods that have been developed lately. These up-to-date methods include water-ligand observed via gradient spectroscopy, isotopic enrichment and paramagnetic labeling of GAG ligands of well-defined chemical structures, characterization of lysine and arginines of GAG-binding sites, dynamics of the GAG-protein complexes, and solid-state NMR for resultant complexes with low solubility. Finally, we discuss how these NMR-based findings of GAG-protein interactions can be used to create molecular models of the complexes to better understand the structure-activity relationship of GAG-protein interactions.15N-labeled proteins and allows for GAG-binding sites in proteins to be identified. The protein atomic chemical shifts of interest are usually obtained through the 15N-edited heteronuclear single quantum coherence (HSQC) spectra of 15N-labeled proteins and medium-sized GAG oligosaccharides whose composition can be heterogeneous. To determine the GAG-binding site, multiple HSQC spectra of the protein with increasing concentrations of the ligand are recorded. These spectra allow for the ligand induced chemical shift changes in protein atoms to be quantified. In particular, the 15N-HSQC experiment measures the chemical shift changes of backbone amide proton and nitrogen (ΔδH and ΔδN) in a residue specific fashion. The magnitudes of the changes are often diagnostic of ligand binding sites as larger changes are interpreted as a consequence of closeness of the atoms to the ligand. But, exceptions can exist. For example, when aromatic rings or charges approach the amide groups, this can sometimes exhibit a larger chemical shift change than expected just from the distances. Nonetheless, the magnitude of CSP for amide 1H and 15N resonances is usually quantified while using the empirical nonlinear Equation (1), where ΔδHN and ΔδN are the observed CSP for 1H and 15N, respectively.Chemical shift perturbation (CSP) or chemical shift mapping is perhaps the mostly used NMR method in GAG-protein interaction studies . It is g1H and 15N in folded proteins  is the total protein concentration, and Δδmax is the maximum GAG-induced chemical shift change for the atom.The weighting factor 0.12 reflects the difference in chemical shift dispersion of proteins . These dAs discussed in 15N-HSQC NMR spectra of hBD6 recorded as a function of the Fx concentration: from a 0 (black) to 4:1 Fx:hBD6 ratio (cyan). NMR titration experiments revealed large changes in hBD6 chemical shifts in a fast exchange equilibrium between free and Fx-bound hBD6, as demonstrated by a gradual shift in peak positions seen by the different 15N-HSQC spectra recorded with increasing concentrations of heparin ligand. The quantitative analysis using the 1HN and 15N chemical shift changes for a subset of hBD6 resonances upon titration with Fx using the above-discussed formulas yielded a Kd value of 4.1 (±2.9) μM  on human beta-defensin 6 (hBD6) mapped by this method is here described . Fx is a±2.9) μM B. Figure15N and 1H resonances significantly affected by the binding of Fx  experiments to complement the CSP methodology. The PRE method is described below at Saturation transfer difference (STD) is a NMR experiment designed to map ligand protons that are involved in the interaction with protein ,23,24,251H reference spectrum of 30 μM of FGF2 and 370 μM of a synthetic heparin octasaccharide composed of only IdoA2S-GlcNS disaccharide units . Crystal structure of FGF2 with heparin showed IdoA2S-GlcNS disaccharide units are essential for strong FGF2-GAG interactions and this was later confirmed by Yu et al. through STD  25]. In. In25]. de units B is showThe bound-state conformation of a ligand, such as a GAG oligosaccharide, can be deduced through a method called exchange-transferred nuclear Overhauser effect (trNOE) . ThroughKd of the complex. To optimize the conditions for the trNOE experiment, parameters such as temperature, ionic strength and protein:GAG oligosaccharide molar ratio must be carefully controlled. The second obstacle can be circumvented while using rotating-frame Overhauser effect (ROE). ROE cross-peaks have different signs depending on whether they are produced by direct or three-spin indirect effects . Th. Th15N-lor GlcA) . Althougor GlcA) . The 15N2,pre is the increase in transverse relaxation rates due to the paramagnetic center, r is the distance between the paramagnetic center and the proton under analysis, γH is the gyromagnetic ratio of proton, gs is the electron g factor for the unpaired electron, ωH is the resonance frequency of the proton under analysis, and τc is the overall rotational correlation time of the whole system. This effect is called PRE and it can be accurately quantified using specific experiments [2 is bigger than Δν1) on nearby atoms, but paramagnetic center with asymmetric g-tensors and favorable relaxation properties can also produce chemical shift displacements (Δδ) in these atoms. These chemical shift changes are referred to as pseudo-contact shifts.Paramagnetically labeled GAG ligands have proven to be useful in structural studies of GAGs’ interactions with several different proteins ,47,48,49eriments ,51. HoweSince the magnitude of PRE is primarily dependent on the distance between the paramagnetic center and nearby atoms, data from this technique are simpler to interpret than CSP, which is also sensitive to allosteric effects. Therefore, PRE is a reliable validation of CSP data. Another key advantage of PRE is that the effective range of the paramagnetic effect can be up to 20 Å. This is ideal for detecting long-range or transient contacts between GAGs and proteins. In several works ,46,47, G15N HSQC spectra of RANTES in the presence of either oxidized or reduced TEMPO-labeled chondroitin sulfate hexasaccharide CS6;4;4 (GlcA-(β1-3)-GalNAc6S-(β1-4)-GlcA-(β1-3)-GalNAc4S-(β1-4)-GlcA(β1-3)-GalNAc4S-TEMPO) are shown in 15N-HSQC spectra, several signals have disappeared upon the oxidation of TEMPO. Those belonging to residues A16 and R21 are highlighted in the upper spectrum. This indicates these residues are in close contact with the paramagetically tagged ligand. Quantitative measurements of the 1H PRE of the amide protons were carried out  is more sensitive at detecting side chain amine signals because of its ability to reduce the relaxation effect of hydrogen exchange on 5N atoms . The Iwaresidues ,56. Theiresidues .We have applied the same methodology to the cytokine pleiotrophin. N-hydroxysuccinimide. Activation of carboxyl groups on these ligands led to amidation reactions between the activated carboxyls on GAGs and nearby lysine side chains on proteins. The conversion of amine to amide eliminated signals of these lysines from the HISQC spectrum, allowing for them to be identified easily. It should be noted that the identity of these modified lysines can also be obtained through mass spectrometry. Because GAG-induced chemical shift changes in lysine side chain signals can be small in many instances, it can be difficult to identify lysines that are involved in GAG binding by direct examination of the HISQC spectrum. Uhrin and co-workers showed that a more sensitive way to identify GAG-binding lysines is through direct cross-linking with GAGs . To do t13C/15N-filtered/edited NOESY experiment used to obtain intermolecular contacts is low in sensitivity as a result of multiple isotopic filters and the need for 13C-labeling. To increase the sensitivity of the NOESY experiments, we have been exploring the use of deuterated protein containing specifically protonated lysines and arginines as a way to improve the sensitivity of the NOESY experiment to intermolecular contacts. By avoiding 13C-labeling, we increase the transverse relaxation time of protons and improve resolution in the both direct and indirect dimensions. However, a lack of 13C-labeling means spectral crowding cannot be resolved with 13C-edited experiments. Fortunately, specifically protonating the amino acids of interest while deuterating other amino acids mostly alleviate the problem. This method of specific labeling has the additional benefit of reducing spin diffusion and longitudinal relaxation, which can further enhance the sensitivity of NOESY to crucial long range intermolecular NOEs [1H homonuclear coupling, leading to additional increases in the signal-to-noise. However, it should be noted that the reduction in longitudinal relaxation requires increases in recycle delay time in order to re-establish the equilibrium magnetization.Detecting intermolecular NOE cross-peaks between protein and GAG is usually quite difficult. Many factors contribute to this, including a lack of non-labile protons at the interface and the often dynamic nature of interactions between protein and GAGs. Compounding the difficulty is that the traditional lar NOEs . It also2H pleiotrophin containing either protonated lysines or arginines. To examine the efficiency and specificity of labeling, we have prepared 1) and transverse (R2) relaxation rates of protein complexes, the total correlation time of the complex can be calculated, and because MW is known to be correlated with the total correlation time, the size of the GAG-induced oligomer can be estimated. The detailed instruction on the procedure can be found on the website of the Northeast Structural Genomics Consortium . In some instances, such as the case of the chemokine CCL5/RANTES, the monomeric and oligomeric species are in slow exchange on the NMR timescale under the right conditions, and therefore can be separately identified in the NMR spectrum  by Kim et al. [15N atoms in the protein backbone in heparin binding sites of VEGF165 while using relaxation dispersion experiments. They noticed that many residues in the GAG-binding sites exhibit dynamic motion in the ms timescale, but these motions were decreased dramatically by the presence of heparin octasaccharide, indicating heparin has stabilized the GAG-binding site. Besides the use of global dynamics in the characterization of GAG-induced oligomerization, quantitative characterization of GAG-induced local changes in protein dynamics can also be done. NMR is an excellent tool for studying protein dynamics at a wide range of timescales. For fast motion on the ps-to-ns timescale in on rates . For slom et al. . They loBorrelia burgdorferi. They found that GAG binding does not change the ps-to-ns timescale motion of DBPB. However, dermatan sulfate oligosaccharides were able to induce slow timescale motion of the residues in the GAG-binding site [15N atom in lysine side chain amine groups. They have been productively applied to the study of DNA-binding proteins [On the other hand, Feng et al. looked at GAG-induced slow timescale motion of decorin-binding protein B (DBPB), a GAG-binding surface adhesin that is found on the Lyme disease bacterium ing site . Interesproteins ,56. Alth13C-enriched heparin octasaccharide, they determined that the octasaccharide remains rigid and is in contact with the amyloid throughout. They also examined amino acids involved in binding heparin by looking at signal dephasing caused by dipolar interactions between 15N atoms in lysine, arginine and histidines, and 13C atoms in the heparin octasaccharide. Surprisingly, the contact between heparin and the amyloid was mediated mostly by histidine residues. Since most of the histidines are located at the junction between peptides in the same cross-sectional layer, they were able to construct a model of these GAG-Aβ40 amyloid complexes. The results also explained the lower heparin affinity of Aβ40 amyloids with two-fold symmetry, since the arrangement of amino acids in two-fold symmetric structures did not allow for correct clustering of the basic amino acids. It should be noted that the availability of 13C-labeled heparin ligands was crucial to their studies. Without this ligand, the important frequency selective rotational echo double resonance experiment used to measure dipole-dipole interactions between 15N and 13C atoms would not be possible.One important biological activity of GAG is their ability to induce formation of amyloids. In fact, GAG is known to promote amyloid formation of a number of proteins, including Aβ40/42, α-synuclein, tau, amylin, and calcitonin ,64,65,661C4 rather than the traditional 4C1 puckering. The inability to consistently specify ring puckers means that the generation of the correct ligand structure using the software is usually cumbersome. The imprecision in the carbohydrate parameters also means longer calculation times are required to find the minimal energy conformation of the ligand in some cases. This makes obtaining ligand structures that are consistent with experimental data difficult. On the other hand, although fully fledged molecular dynamics simulation software usually have more accurate force field and realistic simulation conditions, the relatively large size of most protein-GAG systems means that unrestricted molecular dynamics simulation is not likely to produce accurate structures in a realistic time frame. Obtaining a large quantity of experimental high-resolution structural information on GAG-protein systems is usually not feasible. As a result, constructing of models of protein-GAG complexes cannot be accomplished while using conventional NMR structure determination protocols. This is because the default simulation conditions in these procedures use simplified force-fields in either implicit solvent or vacuum models. Although these parameters greatly expedite the calculation, they require more experimental information to arrive at the correct structure, which is unattainable for most protein-GAG systems. The available force field parameters for creating GAG ligands are also lacking in these software. For instance, molecular topology specification in XPLOR-NIH  and CNS To overcome these limitations, we have been using a hybrid approach to create realistic models of protein-GAG complexes. In particular, we first obtain the coordinate of the GAG ligands while using AMBER and its GLYCAM force field, which has just been updated to include monosaccharides specific to GAGs . This stGAG-dependent regulation of proteins is a pivotal biochemical process in many physiological and pathological events. The understanding of the resultant GAG-protein complexes at the atomic level is necessary to better comprehend the underlying mechanisms that are involved in these events and makes the design of new drugs targeting these proteins possible. NMR spectroscopy is a versatile and robust analytical technique that is often employed in the studies of GAG-protein interactions. Through isotopic labeling, not only is NMR capable of examining the physicochemical properties of the complexes as a whole, but it also excels at examining changes affected by its binding partner in each individual component. Multiple NMR methods are available or under development for assessing the characteristics of the GAG-protein complexes. Here, we reviewed the theoretical and practical aspects of the most common methods that are found in the literature. The experiments discussed herein were CSP, STD, trNOE, PRE, and waterLOGSY. In addition, more recent methods utilized to assess contribution of arginine and lysine side chains in interactions with GAGs and computational approaches commonly employed to model GAG-protein interactions while using NMR-derived information are also presented. We also discussed the importance of measuring spin relaxation rates for assessing dynamic information of the GAG-protein complexes in solution as well as the preparation of GAG oligosaccharides of defined chemical structures for isotopic and paramagenetic labeling so they can be used as functional probes in GAG-protein interaction studies. The number of investigations in this field has increased considerably during the last few years. These methods have been proven to be essential not only to the structural biologists, but also offer benefits to those professionals working in drug discovery. In this regard, we believe that NMR spectroscopy will continue to play a key role in future investigations of GAG-protein interactions.