Adeno-associated virus

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https://en.wikipedia.org/wiki/Adeno-associated_virus 

 

Adeno-associated virus
Adeno-associated virus serotype 2 structure from 1LP3. One fivefold axis shown center.
Scientific classification
(unranked):	Virus
Realm:	Monodnaviria
Kingdom:	Shotokuvirae
Phylum:	Cossaviricota
Class:	Quintoviricetes
Order:	Piccovirales
Family:	Parvoviridae
Subfamily:	Parvovirinae
Genus:	Dependoparvovirus
Viruses included:
Adeno-associated dependoparvovirus A Adeno-associated dependoparvovirus B

Adeno-associated viruses (AAV) are small viruses that infect humans and some other primate species. They belong to the genus Dependoparvovirus, which in turn belongs to the family Parvoviridae. They are small (20 nm) replication-defective, nonenveloped viruses.

AAV are not currently known to cause disease. The viruses cause a very mild immune response. Several additional features make AAV an attractive candidate for creating viral vectors for gene therapy, and for the creation of isogenic human disease models. Gene therapy vectors using AAV can infect both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the genome of the host cell, although in the native virus integration of virally carried genes into the host genome does occur. Integration can be important for certain applications, but can also have unwanted consequences. Recent human clinical trials using AAV for gene therapy in the retina have shown promise.

History

The adeno-associated virus (AAV), previously thought to be a contaminant in adenovirus preparations, was first identified as a dependoparvovirus in the 1960s in the laboratories of Bob Atchison at Pittsburgh and Wallace Rowe at NIH. Serological studies in humans subsequently indicated that, despite being present in people infected by helper viruses such as adenovirus or herpes virus, AAV itself did not cause any disease.

Use in gene therapy

Advantages and drawbacks

Wild-type AAV has attracted considerable interest from gene therapy researchers due to a number of features. Chief amongst these is the virus's apparent lack of pathogenicity. It can also infect non-dividing cells and has the ability to stably integrate into the host cell genome at a specific site (designated AAVS1) in the human chromosome 19. This feature makes it somewhat more predictable than retroviruses, which present the threat of a random insertion and of mutagenesis, which is sometimes followed by development of a cancer. The AAV genome integrates most frequently into the site mentioned, while random incorporations into the genome take place with a negligible frequency. Development of AAVs as gene therapy vectors, however, has eliminated this integrative capacity by removal of the rep and cap from the DNA of the vector. The desired gene together with a promoter to drive transcription of the gene is inserted between the inverted terminal repeats (ITRs) that aid in concatemer formation in the nucleus after the single-stranded vector DNA is converted by host cell DNA polymerase complexes into double-stranded DNA. AAV-based gene therapy vectors form episomal concatemers in the host cell nucleus. In non-dividing cells, these concatemers remain intact for the life of the host cell. In dividing cells, AAV DNA is lost through cell division, since the episomal DNA is not replicated along with the host cell DNA. Random integration of AAV DNA into the host genome is detectable but occurs at very low frequency. AAVs also present very low immunogenicity, seemingly restricted to generation of neutralizing antibodies, while they induce no clearly defined cytotoxic response. This feature, along with the ability to infect quiescent cells present their dominance over adenoviruses as vectors for human gene therapy. 

Use of the virus does present some disadvantages. The cloning capacity of the vector is relatively limited and most therapeutic genes require the complete replacement of the virus's 4.8 kilobase genome. Large genes are, therefore, not suitable for use in a standard AAV vector. Options are currently being explored to overcome the limited coding capacity. The AAV ITRs of two genomes can anneal to form head-to-tail concatemers, almost doubling the capacity of the vector. Insertion of splice sites allows for the removal of the ITRs from the transcript. 

Because of AAV's specialized gene therapy advantages, researchers have created an altered version of AAV termed self-complementary adeno-associated virus (scAAV). Whereas AAV packages a single strand of DNA and must wait for its second strand to be synthesized, scAAV packages two shorter strands that are complementary to each other. By avoiding second-strand synthesis, scAAV can express more quickly, although as a caveat, scAAV can only encode half of the already limited capacity of AAV. Recent reports suggest that scAAV vectors are more immunogenic than single stranded adenovirus vectors, inducing a stronger activation of cytotoxic T lymphocytes.

Humoral immunity instigated by infection with the wild type is thought to be common. The associated neutralising activity limits the usefulness of the most commonly used serotype AAV2 in certain applications. Accordingly, the majority of clinical trials under way involve delivery of AAV2 into the brain, a relatively immunologically privileged organ. In the brain, AAV2 is strongly neuron-specific.

Clinical trials

To date, AAV vectors have been used in over 117 clinical trials worldwide, approximately 5.6% of virus-vectored gene-therapy trials. Recently, promising results have been obtained from Phase 1 and Phase 2 trials for a number of diseases, including Leber's congenital amaurosis, hemophilia, congestive heart failure, spinal muscular atrophy, lipoprotein lipase deficiency, and Parkinson's disease.

Selected clinical trials using AAV-based vectors

Indication	Gene	Route of administration	Phase	Subject number	Status
Cystic fibrosis	CFTR	Lung, via aerosol	I	12	Complete
	CFTR	Lung, via aerosol	II	38	Complete
	CFTR	Lung, via aerosol	II	100	Complete
Hemophilia B	FIX	Intramuscular	I	9	Complete
	FIX	Hepatic artery	I	6	Ended
Arthritis	TNFR:Fc	Intraarticular	I	1	Ongoing
Hereditary emphysema	AAT	Intramuscular	I	12	Ongoing
Leber's congenital amaurosis	RPE65	Subretinal	I–II	Multiple	Several ongoing and complete
Age-related macular degeneration	sFlt-1	Subretinal	I–II	24	Ongoing
Duchenne muscular dystrophy	SGCA	Intramuscular	I	10	Ongoing
Parkinson's disease	GAD65, GAD67	Intracranial	I	12	Complete
Canavan disease	AAC	Intracranial	I	21	Ongoing
Batten disease	CLN2	Intracranial	I	10	Ongoing
Alzheimer's disease	NGF	Intracranial	I	6	Ongoing
Spinal muscular atrophy	SMN1	Intravenous and Intrathecal	I–III	15	Several ongoing and complete
Congestive heart failure	SERCA2a	Intra-coronary	IIb	250	Ongoing

Structure

Two adenovirus particles surrounded by numerous, smaller adeno-associated viruses (negative-staining electron microscopy, magnification approximately 200,000×)

Genome, transcriptome and proteome

The AAV genome is built of single-stranded deoxyribonucleic acid (ssDNA), either positive- or negative-sensed, which is about 4.7 kilobase long. The genome comprises ITRs at both ends of the DNA strand, and two open reading frames (ORFs): rep and cap. The former is composed of four overlapping genes encoding Rep proteins required for the AAV life cycle, and the latter contains overlapping nucleotide sequences of capsid proteins: VP1, VP2 and VP3, which interact to form a capsid with icosahedral symmetry.

ITR sequences

The inverted terminal repeat (ITR) sequences comprise 145 bases each. They were named so because of their symmetry, which was shown to be required for efficient multiplication of the AAV genome. The feature of these sequences that gives them this property is their ability to form a hairpin, which contributes to so-called self-priming that allows primase-independent synthesis of the second DNA strand. The ITRs were also shown to be required for both integration of the AAV DNA into the host cell genome (19th chromosome in humans) and rescue from it, as well as for efficient encapsidation of the AAV DNA combined with generation of a fully assembled, deoxyribonuclease-resistant AAV particles.

With regard to gene therapy, ITRs seem to be the only sequences required in cis next to the therapeutic gene: structural (cap) and packaging (rep) proteins can be delivered in trans. With this assumption many methods were established for efficient production of recombinant AAV (rAAV) vectors containing a reporter or therapeutic gene. However, it was also published that the ITRs are not the only elements required in cis for the effective replication and encapsidation. A few research groups have identified a sequence designated cis-acting Rep-dependent element (CARE) inside the coding sequence of the rep gene. CARE was shown to augment the replication and encapsidation when present in cis.

rep gene and Rep proteins

On the "left side" of the genome there are two promoters called p5 and p19, from which two overlapping messenger ribonucleic acids (mRNAs) of different length can be produced. Each of these contains an intron which can be either spliced out or not. Given these possibilities, four various mRNAs, and consequently four various Rep proteins with overlapping sequence can be synthesized. Their names depict their sizes in kilodaltons (kDa): Rep78, Rep68, Rep52 and Rep40. Rep78 and 68 can specifically bind the hairpin formed by the ITR in the self-priming act and cleave at a specific region, designated terminal resolution site, within the hairpin. They were also shown to be necessary for the AAVS1-specific integration of the AAV genome. All four Rep proteins were shown to bind ATP and to possess helicase activity. It was also shown that they upregulate the transcription from the p40 promoter (mentioned below), but downregulate both p5 and p19 promoters.

cap gene and VP proteins

The right side of a positive-sensed AAV genome encodes overlapping sequences of three capsid proteins, VP1, VP2 and VP3, which start from one promoter, designated p40. The molecular weights of these proteins are 87, 72 and 62 kiloDaltons, respectively. The AAV capsid is composed of a mixture of VP1, VP2, and VP3 totaling 60 monomers arranged in icosahedral symmetry in a ratio of 1:1:10, with an estimated size of 3.9 MegaDaltons. The crystal structure of the VP3 protein was determined by Xie, Bue, et al. 

AAV2 capsid, shown as a ribbon diagram, with the back half hidden for clarity. One fivefold symmetry axis is shown center.

 

The cap gene produces an additional, non-structural protein called the Assembly-Activating Protein (AAP). This protein is produced from ORF2 and is essential for the capsid-assembly process. The exact function of this protein in the assembly process and its structure have not been solved to date.

All three VPs are translated from one mRNA. After this mRNA is synthesized, it can be spliced in two different manners: either a longer or shorter intron can be excised resulting in the formation of two pools of mRNAs: a 2.3 kb- and a 2.6 kb-long mRNA pool. Usually, especially in the presence of adenovirus, the longer intron is preferred, so the 2.3-kb-long mRNA represents the so-called "major splice". In this form the first AUG codon, from which the synthesis of VP1 protein starts, is cut out, resulting in a reduced overall level of VP1 protein synthesis. The first AUG codon that remains in the major splice is the initiation codon for VP3 protein. However, upstream of that codon in the same open reading frame lies an ACG sequence (encoding threonine) which is surrounded by an optimal Kozak context. This contributes to a low level of synthesis of VP2 protein, which is actually VP3 protein with additional N terminal residues, as is VP1.

Since the bigger intron is preferred to be spliced out, and since in the major splice the ACG codon is a much weaker translation initiation signal, the ratio at which the AAV structural proteins are synthesized in vivo is about 1:1:20, which is the same as in the mature virus particle. The unique fragment at the N terminus of VP1 protein was shown to possess the phospholipase A2 (PLA2) activity, which is probably required for the releasing of AAV particles from late endosomes. Muralidhar et al. reported that VP2 and VP3 are crucial for correct virion assembly. More recently, however, Warrington et al. showed VP2 to be unnecessary for the complete virus particle formation and an efficient infectivity, and also presented that VP2 can tolerate large insertions in its N terminus, while VP1 can not, probably because of the PLA2 domain presence.

Classification, serotypes, receptors and native tropism

Two species of AAV were recognised by the International Committee on Taxonomy of Viruses in 2013: adeno-associated dependoparvovirus A (formerly AAV-1, -2, -3 and -4) and adeno-associated dependoparvovirus B (formerly AAV-5).

Until the 1990s, virtually all AAV biology was studied using AAV serotype 2. However, AAV is highly prevalent in humans and other primates and several serotypes have been isolated from various tissue samples. Serotypes 2, 3, 5, and 6 were discovered in human cells, AAV serotypes 1, 4, and 7–11 in nonhuman primate samples. As of 2006 there have been 11 AAV serotypes described, the 11th in 2004. AAV capsid proteins contain 12 hypervariable surface regions, with most variability occurring in the threefold proximal peaks, but the parvovirus genome in general presents highly conserved replication and structural genes across serotypes. All of the known serotypes can infect cells from multiple diverse tissue types. Tissue specificity is determined by the capsid serotype and pseudotyping of AAV vectors to alter their tropism range will likely be important to their use in therapy.

Serotype 2

Serotype 2 (AAV2) has been the most extensively examined so far. AAV2 presents natural tropism towards skeletal muscles, neurons, vascular smooth muscle cells and hepatocytes.

Three cell receptors have been described for AAV2: heparan sulfate proteoglycan (HSPG), a_Vβ₅ integrin and fibroblast growth factor receptor 1 (FGFR-1). The first functions as a primary receptor, while the latter two have a co-receptor activity and enable AAV to enter the cell by receptor-mediated endocytosis. These study results have been disputed by Qiu, Handa, et al. HSPG functions as the primary receptor, though its abundance in the extracellular matrix can scavenge AAV particles and impair the infection efficiency.

Studies have shown that serotype 2 of the virus (AAV-2) apparently kills cancer cells without harming healthy ones. "Our results suggest that adeno-associated virus type 2, which infects the majority of the population but has no known ill effects, kills multiple types of cancer cells yet has no effect on healthy cells," said Craig Meyers, a professor of immunology and microbiology at the Penn State College of Medicine in Pennsylvania in 2005. This could lead to a new anti-cancer agent.

Other serotypes

Although AAV2 is the most popular serotype in various AAV-based research, it has been shown that other serotypes can be more effective as gene delivery vectors. For instance AAV6 appears much better in infecting airway epithelial cells, AAV7 presents very high transduction rate of murine skeletal muscle cells (similar to AAV1 and AAV5), AAV8 is superb in transducing hepatocytes and AAV1 and 5 were shown to be very efficient in gene delivery to vascular endothelial cells. In the brain, most AAV serotypes show neuronal tropism, while AAV5 also transduces astrocytes. AAV6, a hybrid of AAV1 and AAV2, also shows lower immunogenicity than AAV2.

Serotypes can differ with the respect to the receptors they are bound to. For example, AAV4 and AAV5 transduction can be inhibited by soluble sialic acids (of different form for each of these serotypes), and AAV5 was shown to enter cells via the platelet-derived growth factor receptor.

Synthetic serotypes

There have been many efforts to engineer and improve new AAV variants for both clinical and research purposes. Such modifications include new tropisms to target specific tissues, and modified surface residues to evade detection by the immune system. Beyond opting for particular strains of recombinant AAV (rAAV) to target particular cells, researchers have also explored AAV pseudotyping, the practice of creating hybrids of certain AAV strains to approach an even more refined target. The hybrid is created by taking a capsid from one strain and the genome from another strain. For example, research involving AAV2/5, a hybrid with the genome of AAV2 and the capsid of AAV5, was able to achieve more accuracy and range in brain cells than AAV2 would be able to achieve unhybridized. Researchers have continued to experiment with pseudotyping by creating strains with hybrid capsids. AAV-DJ has a hybrid capsid from eight different strains of AAV; as such, it can infect different cells throughout many areas of the body, a property which a single strain of AAV with a limited tropism would not have. Other efforts to engineer and improve new AAV variants have involved the ancestral reconstruction of virus variants to generate new vectors with enhanced properties for clinical applications and the study of AAV biology.

Immunology

AAV is of particular interest to gene therapists due to its apparent limited capacity to induce immune responses in humans, a factor which should positively influence vector transduction efficiency while reducing the risk of any immune-associated pathology. 

AAV is not considered to have any known role in disease.

Innate

The innate immune response to the AAV vectors has been characterised in animal models. Intravenous administration in mice causes transient production of pro-inflammatory cytokines and some infiltration of neutrophils and other leukocytes into the liver, which seems to sequester a large percentage of the injected viral particles. Both soluble factor levels and cell infiltration appear to return to baseline within six hours. By contrast, more aggressive viruses produce innate responses lasting 24 hours or longer.

Humoral

The virus is known to instigate robust humoral immunity in animal models and in the human population, where up to 80% of individuals are thought to be seropositive for AAV2. Antibodies are known to be neutralising, and for gene therapy applications these do impact on vector transduction efficiency via some routes of administration. As well as persistent AAV specific antibody levels, it appears from both prime-boost studies in animals and from clinical trials that the B-cell memory is also strong. In seropositive humans, circulating IgG antibodies for AAV2 appear to be primarily composed of the IgG1 and IgG2 subclasses, with little or no IgG3 or IgG4 present.

Cell-mediated

The cell-mediated response to the virus and to vectors is poorly characterised, and has been largely ignored in the literature as recently as 2005. Clinical trials using an AAV2-based vector to treat haemophilia B seem to indicate that targeted destruction of transduced cells may be occurring. Combined with data that shows that CD8+ T-cells can recognise elements of the AAV capsid in vitro, it appears that there may be a cytotoxic T lymphocyte response to AAV vectors. Cytotoxic responses would imply the involvement of CD4+ T helper cells in the response to AAV and in vitro data from human studies suggests that the virus may indeed induce such responses, including both Th1 and Th2 memory responses. A number of candidate T cell stimulating epitopes have been identified within the AAV capsid protein VP1, which may be attractive targets for modification of the capsid if the virus is to be used as a vector for gene therapy.

Infection cycle

There are several steps in the AAV infection cycle, from infecting a cell to producing new infectious particles:

1. attachment to the cell membrane
2. receptor-mediated endocytosis
3. endosomal trafficking
4. escape from the late endosome or lysosome
5. translocation to the nucleus
6. uncoating
7. formation of double-stranded DNA replicative form of the AAV genome
8. expression of rep genes
9. genome replication
10. expression of cap genes, synthesis of progeny ssDNA particles
11. assembly of complete virions, and
12. release from the infected cell.

Some of these steps may look different in various types of cells, which, in part, contributes to the defined and quite limited native tropism of AAV. Replication of the virus can also vary in one cell type, depending on the cell's current cell cycle phase.

The characteristic feature of the adeno-associated virus is a deficiency in replication and thus its inability to multiply in unaffected cells. Adeno-associated virus spreads by co-infecting a cell with a helper virus. The first helper virus that was described as providing successful generation of new AAV particles, was the adenovirus, from which the AAV name originated. It was then shown that AAV replication can be facilitated by selected proteins derived from the adenovirus genome, by other viruses such as HSV or vaccinia, or by genotoxic agents, such as UV irradiation or hydroxyurea. Depending on the presence or absence of a helper virus, the life cycle of AAV follows either a lytic or lysogenic pathway, respectively. If there is a helper virus, AAV's gene expression activates, allowing the virus to replicate using the host cell's polymerase. When the helper virus kills the host cell, the new AAV virions are released. If there is not a helper virus present, AAV exhibits lysogenic behavior. When AAV infects a cell alone, its gene expression is repressed (AAV does not replicate), and its genome is incorporated into the host genome (into human chromosome 19). In rare cases, lysis can occur without a helper virus, but usually AAV can not replicate and kill a cell on its own.

The minimal set of the adenoviral genes required for efficient generation, of progeny AAV particles, was discovered by Matsushita, Ellinger et al. This discovery allowed for new production methods of recombinant AAV, which do not require adenoviral co-infection of the AAV-producing cells. In the absence of helper virus or genotoxic factors, AAV DNA can either integrate into the host genome or persist in episomal form. In the former case integration is mediated by Rep78 and Rep68 proteins and requires the presence of ITRs flanking the region being integrated. In mice, the AAV genome has been observed persisting for long periods of time in quiescent tissues, such as skeletal muscles, in episomal form (a circular head-to-tail conformation).

Gene therapy of the human retina

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Gene_therapy_of_the_human_retina 

 

Retinal gene therapy holds a promise in treating different forms of non-inherited and inherited blindness.

In 2008, three independent research groups reported that patients with the rare genetic retinal disease Leber's congenital amaurosis had been successfully treated using gene therapy with adeno-associated virus (AAV). In all three studies, an AAV vector was used to deliver a functional copy of the RPE65 gene, which restored vision in children suffering from LCA. These results were widely seen as a success in the gene therapy field, and have generated excitement and momentum for AAV-mediated applications in retinal disease.

In retinal gene therapy, the most widely used vectors for ocular gene delivery are based on adeno-associated virus. The great advantage in using adeno-associated virus for the gene therapy is that it poses minimal immune responses and mediates long-term transgene expression in a variety of retinal cell types. For example, tight junctions that form the blood-retina barrier, separate subretinal space from the blood supply, providing protection from microbes and decreasing most immune-mediated damages.

Clinical trials

Leber's congenital amaurosis

Preclinical studies in mouse models of Leber's congenital amaurosis (LCA) were published in 1996 and a study in dogs published in 2001. In 2008, three groups reported results of clinical trials using adeno-associated virus for LCA. In these studies, an AAV vector encoding the RPE65 gene was delivered via a "subretinal injection", where a small amount of fluid is injected underneath the retina in a short surgical procedure. Development continued, and in December 2017 the FDA approved Voretigene neparvovec (Luxturna), an adeno-associated virus vector-based gene therapy for children and adults with biallelic RPE65 gene mutations responsible for retinal dystrophy, including Leber congenital amaurosis. People must have viable retinal cells as a prerequisite for the intraocular administration of the drug.

Age-related macular degeneration

Following the successful clinical trials in LCA, researchers have been developing similar treatments using adeno-associated virus for age-related macular degeneration (AMD). To date, efforts have focused on long-term delivery of VEGF inhibitors to treat the wet form of macular degeneration. Whereas wet AMD is currently treated using frequent injections of recombinant protein into the eyeball, the goal of these treatments is long-term disease management following a single administration. One such study is being conducted at the Lions Eye Institute in Australia in collaboration with Avalanche Biotechnologies, a US-based biotechnology start-up. Another early-stage study is sponsored by Genzyme Corporation.

Choroideremia

In October 2011, the first clinical trial was announced for the treatment of choroideremia. Dr. Robert MacLaren of the University of Oxford, who lead the trial, co-developed the treatment with Dr. Miguel Seabra of the Imperial College, London. This Phase 1/2 trial used subretinal AAV to restore the REP gene in affected patients. Initial results of the trial were reported in January 2014 as promising as all six patients had better vision.

Color blindness

Recent research has shown that AAV can successfully restore color vision to treat color blindness in adult monkeys. Although this treatment has not yet entered clinical trials for humans, this work was considered a breakthrough for the ability to target cone photoreceptors.

Mechanism

Physiological components in retinal gene therapy

The vertebrate neural retina composed of several layers and distinct cell types. A number of these cell types are implicated in retinal diseases, including retinal ganglion cells, which degenerate in glaucoma, the rod and cone photoreceptors, which are responsive to light and degenerate in retinitis pigmentosa, macular degeneration, and other retinal diseases, and the retinal pigment epithelium (RPE), which supports the photoreceptors and is also implicated in retinitis pigmentosa and macular degeneration. 

In retinal gene therapy, AAV is capable of "transducing" these various cell types by entering the cells and expressing the therapeutic DNA sequence. Since the cells of the retina are non-dividing, AAV continues to persist and provide expression of the therapeutic DNA sequence over a long time period that can last several years.

AAV tropism and routes of administration

AAV is capable of transducing multiple cell types within the retina. AAV serotype 2, the most well-studied type of AAV, is commonly administered in one of two routes: intravitreal or subretinal. Using the intravitreal route, AAV is injected in the vitreous humor of the eye. Using the subretinal route, AAV is injected underneath the retina, taking advantage of the potential space between the photoreceptors and RPE layer, in a short surgical procedure. Although this is more invasive than the intravitreal route, the fluid is absorbed by the RPE and the retina flattens in less than 14 hours without complications. Intravitreal AAV targets retinal ganglion cells and a few Muller glial cells. Subretinal AAV efficiently targets photoreceptors and RPE cells.

The reason that different routes of administration lead to different cell types being transfected (e.g., different tropism) is that the inner limiting membrane (ILM) and the various retinal layers act as physical barriers for the delivery of drugs and vectors to the deeper retinal layers. Thus overall, subretinal AAV is 5-10 times more efficient than delivery using the intravitreal route.

Tropism modification and novel AAV vectors

One important factor in gene delivery is developing altered cell tropisms to narrow or broaden rAAV-mediated gene delivery and to increase its efficiency in tissues. Specific properties like capsid conformation, cell targeting strategies can determine which cell types are affected and also the efficiency of the gene transfer process. Different kinds of modification can be undertaken. For example, modification by chemical, immunological or genetic changes that enables the AAV2 capsid to interact with specific cell surface molecules.

Initial studies with AAV in the retina have utilized AAV serotype 2. Researchers are now beginning to develop new variants of AAV, based on naturally-occurring AAV serotypes and engineered AAV variants.

Several naturally-occurring serotypes of AAV have been isolated that can transduce retinal cells. Following intravitreal injection, only AAV serotypes 2 and 8 were capable of transducing retinal ganglion cells. Occasional Muller cells were transduced by AAV serotypes 2, 8, and 9. Following subretinal injection, serotypes 2, 5, 7, and 8 efficiently transduced photoreceptors, and serotypes 1, 2, 5, 7, 8, and 9 efficiently transduce RPE cells.

One example of an engineered variant has recently been described that efficiently transduces Muller glia following intravitreal injection, and has been used to rescue an animal model of aggressive, autosomal-dominant retinitis pigmentosa.

AAV and immune privilege in the retina

Importantly, the retina is immune-privileged, and thus does not experience a significant inflammation or immune-response when AAV is injected. Immune response to gene therapy vectors is what has caused previous attempts at gene therapy to fail, and is considered a key advantage of gene therapy in the eye. Re-administration has been successful in large animals, indicating that no long-lasting immune response is mounted.

Recent data indicates that the subretinal route may be subject to a greater degree of immune privilege compared to the intravitreal route.

Promoter sequence

Expression in various retinal cell types can be determined by the promoter sequence. In order to restrict expression to a specific cell type, a tissue-specific or cell-type specific promoter can be used.

For example, in rats the murine rhodopsin gene drive the expression in AAV2, GFP reporter product was found only in rat photoreceptors, not in any other retinal cell type or in the adjacent RPE after subretinal injection. On the other hand, if ubiquitously expressed immediate-early cytomegalovirus (CMV) enhancer-promoter is expressed in a wide variety of transfected cell types. Other ubiquitous promoters such as the CBA promoter, a fusion of the chicken-actin promoter and CMV immediate-early enhancer, allows stable GFP reporter expression in both RPE and photoreceptor cells after subretinal injections.

Modulation of expression

Sometimes modulation of transgene expression may be necessary since strong constitutive expression of a therapeutic gene in retinal tissues could be deleterious for long-term retinal function. Different methods have been utilized for the expression modulation. One way is using exogenously regulatable promoter system in AAV vectors. For example, the tetracycline-inducible expression system uses a silencer/transactivator AAV2 vector and a separate inducible doxycycline-responsive coinjection. When induction occurs by oral doxycycline, this system shows tight regulation of gene expression in both photoreceptor and RPE cells.

Examples and animal models

Targeting RPE

One study that was done by Royal College of Surgeons (RCS) in rat model shows that a recessive mutation in a receptor tyrposine kinase gene, mertk results in a premature stop codon and impaired phagocytosis function by RPE cells. This mutation causes the accumulation of outer segment debris in the subretinal space, which causes photoreceptor cell death. The model organism with this disease received a subretinal injection of AAV serotype 2 carrying a mouse Mertk cDNA under the control of either the CMV or RPE65 promoters. This treatment was found to prolong photoreceptor cell survival for several months and also the number of photoreceptor was 2.5 fold higher in AAV-Mertk- treated eyes compared with controls 9 weeks after injection, also they found decreased amount of debris in the subretinal space.

The protein RPE65 is used in the retinoid cycle where the all-trans-retinol within the rod outer segment is isomerized to its 11-cis form and oxidized to 11-cis retinal before it goes back to the photoreceptor and joins with opsin molecule to form functional rhodopsin. In animal knockout model (RPE65-/-), gene transfer experiment shows that early intraocular delivery of human RPE65 vector on embryonic day 14 shows efficient transduction of retinal pigment epithelium in the RPE65-/- knockout mice and rescues visual functions. This shows successful gene therapy can be attributed to early intraocular deliver to the diseased animal.

Targeting of photoreceptors

Juvenile retinoschisis is a disease that affects the nerve tissue in the eye. This disease is an X-linked recessive degenerative disease of the central macula region, and it is caused by mutation in the RSI gene encoding the protein retinoschisin. Retinoschisin is produced in the photoreceptor and bipolar cells and it is critical in maintaining the synaptic integrity of the retina.

Specifically the AAV 5 vector containing the wild-type human RSI cDNA driven by a mouse opsin promoter showed long-term retinal functional and structural recovery. Also the retinal structural reliability improved greatly after the treatment, characterized by an increase in the outer nuclear layer thickness.

Retinitis pigmentosa

Retinitis pigmentosa is an inherited disease which leads to progressive night blindness and loss of peripheral vision as a result of photoreceptor cell death. Most people who suffer from RP are born with rod cells that are either dead or dysfunctional, so they are effectively blind at nighttime, since these are the cells responsible for vision in low levels of light. What follows often is the death of cone cells, responsible for color vision and acuity, at light levels present during the day. Loss of cones leads to full blindness as early as five years old, but may not onset until many years later. There have been multiple hypotheses about how the lack of rod cells can lead to the death of cone cells. Pinpointing a mechanism for RP is difficult because there are more than 39 genetic loci and genes correlated with this disease. In an effort to find the cause of RP, there have been different gene therapy techniques applied to address each of the hypotheses.

Different types of inheritance can attribute to this disease; autosomal recessive, autosomal dominant, X-linked type, etc. The main function of rhodopsin is initiating the phototransduction cascade. The opsin proteins are made in the photoreceptor inner segments, then transported to the outer segment, and eventually phagocytized by the RPE cells. When mutations occur in the rhodopsin the directional protein movement is affected because the mutations can affect protein folding, stability, and intracellular trafficking. One approach is introducing AAV-delivered ribozymes designed to target and destroy a mutant mRNA.

The way this system operates was shown in animal model that have a mutant rhodopsin gene. The injected AAV-ribozymes were optimized in vitro and used to cleave the mutant mRNA transcript of P23H (where most mutation occur) in vivo.

Another mutation in the rhodopsin structural protein, specifically peripherin 2 which is a membrane glycoprotein involved in the formation of photoreceptor outersegment disk, can lead to recessive RP and macular degeneration in human (19). In a mouse experiment, AAV2 carrying a wild-type peripherin 2 gene driven by a rhodopsin promoter was delivered to the mice by subretinal injection. The result showed improvement in photoreceptor structure and function which was detected by ERG (electroretinogram). The result showed improvement of photoreceptor structure and function which was detected by ERG. Also peripherin 2 was detected at the outer segment layer of the retina 2 weeks after injection and therapeutic effects were noted as soon as 3 weeks after injection. A well-defined outer segment containing both peripherin2 and rhodopsin was present 9-month after injection.

Since apoptosis can be the cause of photoreceptor death in most of the retinal dystrophies. It has been known that survival factors and antiapoptoic reagents can be an alternative treatment if the mutation is unknown for gene replacement therapy. Some scientists have experimented with treating this issue by injecting substitute trophic factors into the eye. One group of researchers injected the rod derived cone viability factor (RdCVF) protein (encoded for by the Nxnl1 (Txnl6) gene) into the eye of the most commonly occurring dominant RP mutation rat models. This treatment demonstrated success in promoting the survival of cone activity, but the treatment served even more significantly to prevent progression of the disease by increasing the actual function of the cones. Experiments were also carried out to study whether supplying AAV2 vectors with cDNA for glial cell line-derived neurotrophic factor (GDNF) can have an anti-apoptosis effect on the rod cells. In looking at an animal model, the opsin transgene contains a truncated protein lacking the last 15 amino acids of the C terminus, which causes alteration in rhodopsin transport to the outer segment and leads to retinal degeneration. When the AAV2-CBA-GDNF vector is administered to the subretinal space, photoreceptor stabilized and rod photoreceptors increased and this was seen in the improved function of the ERG analysis. Successful experiments in animals have also been carried out using ciliary neurotrophic factor (CNTF), and CNTF is currently being used as a treatment in human clinical trials.

AAV-based treatment for retinal neovascular diseases

Ocular neovascularization (NV) is the abnormal formation of new capillaries from already existing blood vessels in the eye, and this is a characteristics for ocular diseases such as diabetic retinopathy (DR), retinopathy of prematurity (ROP) and (wet form) age-related macular degeneration (AMD). One of the main players in these diseases is VEGF (Vascular endothelial growth factor) which is known to induce vessel leakage and which is also known to be angiogenic. In normal tissues VEGF stimulates endothelial cell proliferation in a dose dependent manner, but such activity is lost with other angiogenic factors.

Many angiostatic factors have been shown to counteract the effect of increasing local VEGF. The naturally occurring form of soluble Flt-1 has been shown to reverse neovascularization in rats, mice, and monkeys.

Pigment epithelium-derived factor (PEDF) also acts as an inhibitor of angiogenesis. The secretion of PEDF is noticeably decreased under hypoxic conditions allowing the endothelial mitogenic activity of VEGF to dominate, suggesting that the loss of PEDF plays a central role in the development of ischemia-driven NV. One clinical finding shows that the levels of PEDF in aqueous humor of human are decreased with increasing age, indicating that the reduction may lead to the development of AMD. In animal model, an AAV with human PEDF cDNA under the control of the CMV promoter prevented choroidal and retinal NV(24). 

The finding suggests that the AAV-mediated expression of angiostatic factors can be implemented to treat NV. This approach could be useful as an alternative to frequent injections of recombinant protein into the eye. In addition, PEDF and sFlt-1 may be able to diffuse through sclera tissue, allowing for the potential to be relatively independent of the intraocular site of administration.

Biometrics

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Biometrics 

At Walt Disney World in Lake Buena Vista, Florida, biometric measurements are taken from the fingers of guests to ensure that a ticket is used by the same person from day to day

 

Biometrics is the technical term for body measurements and calculations. It refers to metrics related to human characteristics. Biometrics authentication (or realistic authentication) is used in computer science as a form of identification and access control. It is also used to identify individuals in groups that are under surveillance.

Biometric identifiers are the distinctive, measurable characteristics used to label and describe individuals. Biometric identifiers are often categorized as physiological versus behavioral characteristics. Physiological characteristics are related to the shape of the body. Examples include, but are not limited to fingerprint, palm veins, facerecognition, DNA, palmprint, handgeometry, irisrecognition, retina and odour/scent. Behavioral characteristics are related to the pattern of behavior of a person, including but not limited to typing rhythm, gait, and voice. Some researchers have coined the term behaviometrics to describe the latter class of biometrics.

More traditional means of access control include token-based identification systems, such as a driver's license or passport, and knowledge-based identification systems, such as a password or personal identification number. Since biometric identifiers are unique to individuals, they are more reliable in verifying identity than token and knowledge-based methods; however, the collection of biometric identifiers raises privacy concerns about the ultimate use of this information.

Biometric functionality

Many different aspects of human physiology, chemistry or behavior can be used for biometric authentication. The selection of a particular biometric for use in a specific application involves a weighting of several factors. Jain et al. (1999) identified seven such factors to be used when assessing the suitability of any trait for use in biometric authentication.

• Universality means that every person using a system should possess the trait.
• Uniqueness means the trait should be sufficiently different for individuals in the relevant population such that they can be distinguished from one another.
• Permanence relates to the manner in which a trait varies over time. More specifically, a trait with 'good' permanence will be reasonably invariant over time with respect to the specific matching algorithm.
• Measurability (collectability) relates to the ease of acquisition or measurement of the trait. In addition, acquired data should be in a form that permits subsequent processing and extraction of the relevant feature sets.
• Performance relates to the accuracy, speed, and robustness of technology used (see performance section for more details).
• Acceptability relates to how well individuals in the relevant population accept the technology such that they are willing to have their biometric trait captured and assessed.
• Circumvention relates to the ease with which a trait might be imitated using an artifact or substitute.

Proper biometric use is very application dependent. Certain biometrics will be better than others based on the required levels of convenience and security. No single biometric will meet all the requirements of every possible application.

The block diagram illustrates the two basic modes of a biometric system. First, in verification (or authentication) mode the system performs a one-to-one comparison of a captured biometric with a specific template stored in a biometric database in order to verify the individual is the person they claim to be. Three steps are involved in the verification of a person. In the first step, reference models for all the users are generated and stored in the model database. In the second step, some samples are matched with reference models to generate the genuine and impostor scores and calculate the threshold. The third step is the testing step. This process may use a smart card, username or ID number (e.g. PIN) to indicate which template should be used for comparison. 'Positive recognition' is a common use of the verification mode, "where the aim is to prevent multiple people from using the same identity".

Second, in identification mode the system performs a one-to-many comparison against a biometric database in an attempt to establish the identity of an unknown individual. The system will succeed in identifying the individual if the comparison of the biometric sample to a template in the database falls within a previously set threshold. Identification mode can be used either for 'positive recognition' (so that the user does not have to provide any information about the template to be used) or for 'negative recognition' of the person "where the system establishes whether the person is who she (implicitly or explicitly) denies to be". The latter function can only be achieved through biometrics since other methods of personal recognition such as passwords, PINs or keys are ineffective.

The first time an individual uses a biometric system is called enrollment. During enrollment, biometric information from an individual is captured and stored. In subsequent uses, biometric information is detected and compared with the information stored at the time of enrollment. Note that it is crucial that storage and retrieval of such systems themselves be secure if the biometric system is to be robust. Biometric data is stored and processed with database servers, encrypted tokens, or physical tokens, while more secure devices will use on-device storage of biometric templates. This type of storage ensures identity authentication without the transference of any sensitive biometric information over the internet to a different server or location. The first block (sensor) is the interface between the real world and the system; it has to acquire all the necessary data. Most of the times it is an image acquisition system, but it can change according to the characteristics desired. The second block performs all the necessary pre-processing: it has to remove artifacts from the sensor, to enhance the input (e.g. removing background noise), to use some kind of normalization, etc. In the third block, necessary features are extracted. This step is an important step as the correct features need to be extracted in an optimal way. A vector of numbers or an image with particular properties is used to create a template. A template is a synthesis of the relevant characteristics extracted from the source. Elements of the biometric measurement that are not used in the comparison algorithm are discarded in the template to reduce the filesize and to protect the identity of the enrollee. However, depending on the scope of the biometric system, original biometric image sources may be retained such as the PIV-cards used in the Federal Information Processing Standard Personal Identity Verification (PIV) of Federal Employees and Contractors (FIPS 201).

During the enrollment phase, the template is simply stored somewhere (on a card or within a database or both). During the matching phase, the obtained template is passed to a matcher that compares it with other existing templates, estimating the distance between them using any algorithm (e.g. Hamming distance). The matching program will analyze the template with the input. This will then be output for a specified use or purpose (e.g. entrance in a restricted area), though it is a fear that the use of biometric data may face mission creep. Selection of biometrics in any practical application depending upon the characteristic measurements and user requirements. In selecting a particular biometric, factors to consider include, performance, social acceptability, ease of circumvention and/or spoofing, robustness, population coverage, size of equipment needed and identity theft deterrence. The selection of a biometric is based on user requirements and considers sensor and device availability, computational time and reliability, cost, sensor size, and power consumption.

Multimodal biometric system

Multimodal biometric systems use multiple sensors or biometrics to overcome the limitations of unimodal biometric systems. For instance iris recognition systems can be compromised by aging irises and electronic fingerprint recognition can be worsened by worn-out or cut fingerprints. While unimodal biometric systems are limited by the integrity of their identifier, it is unlikely that several unimodal systems will suffer from identical limitations. Multimodal biometric systems can obtain sets of information from the same marker (i.e., multiple images of an iris, or scans of the same finger) or information from different biometrics (requiring fingerprint scans and, using voice recognition, a spoken passcode).

Multimodal biometric systems can fuse these unimodal systems sequentially, simultaneously, a combination thereof, or in series, which refer to sequential, parallel, hierarchical and serial integration modes, respectively. Fusion of the biometrics information can occur at different stages of a recognition system. In case of feature level fusion, the data itself or the features extracted from multiple biometrics are fused. Matching-score level fusion consolidates the scores generated by multiple classifiers pertaining to different modalities. Finally, in case of decision level fusion the final results of multiple classifiers are combined via techniques such as majority voting. Feature level fusion is believed to be more effective than the other levels of fusion because the feature set contains richer information about the input biometric data than the matching score or the output decision of a classifier. Therefore, fusion at the feature level is expected to provide better recognition results.

Spoof attacks consist in submitting fake biometric traits to biometric systems, and are a major threat that can curtail their security. Multi-modal biometric systems are commonly believed to be intrinsically more robust to spoof attacks, but recent studies have shown that they can be evaded by spoofing even a single biometric trait.

Performance

The following are used as performance metrics for biometric systems:

• False match rate (FMR, also called FAR = False Accept Rate): the probability that the system incorrectly matches the input pattern to a non-matching template in the database. It measures the percent of invalid inputs that are incorrectly accepted. In case of similarity scale, if the person is an imposter in reality, but the matching score is higher than the threshold, then he is treated as genuine. This increases the FMR, which thus also depends upon the threshold value.
• False non-match rate (FNMR, also called FRR = False Reject Rate): the probability that the system fails to detect a match between the input pattern and a matching template in the database. It measures the percent of valid inputs that are incorrectly rejected.
• Receiver operating characteristic or relative operating characteristic (ROC): The ROC plot is a visual characterization of the trade-off between the FMR and the FNMR. In general, the matching algorithm performs a decision based on a threshold that determines how close to a template the input needs to be for it to be considered a match. If the threshold is reduced, there will be fewer false non-matches but more false accepts. Conversely, a higher threshold will reduce the FMR but increase the FNMR. A common variation is the Detection error trade-off (DET), which is obtained using normal deviation scales on both axes. This more linear graph illuminates the differences for higher performances (rarer errors).
• Equal error rate or crossover error rate (EER or CER): the rate at which both acceptance and rejection errors are equal. The value of the EER can be easily obtained from the ROC curve. The EER is a quick way to compare the accuracy of devices with different ROC curves. In general, the device with the lowest EER is the most accurate.
• Failure to enroll rate (FTE or FER): the rate at which attempts to create a template from an input is unsuccessful. This is most commonly caused by low-quality inputs.
• Failure to capture rate (FTC): Within automatic systems, the probability that the system fails to detect a biometric input when presented correctly.
• Template capacity: the maximum number of sets of data that can be stored in the system.

History

An early cataloguing of fingerprints dates back to 1881 when Juan Vucetich started a collection of fingerprints of criminals in Argentina. Josh Ellenbogen and Nitzan Lebovic argued that Biometrics originated in the identification systems of criminal activity developed by Alphonse Bertillon (1853–1914) and by Francis Galton's theory of fingerprints and physiognomy. According to Lebovic, Galton's work "led to the application of mathematical models to fingerprints, phrenology, and facial characteristics", as part of "absolute identification" and "a key to both inclusion and exclusion" of populations. Accordingly, "the biometric system is the absolute political weapon of our era" and a form of "soft control". The theoretician David Lyon showed that during the past two decades biometric systems have penetrated the civilian market, and blurred the lines between governmental forms of control and private corporate control. Kelly A. Gates identified 9/11 as the turning point for the cultural language of our present: "in the language of cultural studies, the aftermath of 9/11 was a moment of articulation, where objects or events that have no necessary connection come together and a new discourse formation is established: automated facial recognition as a homeland security technology."

Adaptive biometric systems

Adaptive biometric systems aim to auto-update the templates or model to the intra-class variation of the operational data. The two-fold advantages of these systems are solving the problem of limited training data and tracking the temporal variations of the input data through adaptation. Recently, adaptive biometrics have received a significant attention from the research community. This research direction is expected to gain momentum because of their key promulgated advantages. First, with an adaptive biometric system, one no longer needs to collect a large number of biometric samples during the enrollment process. Second, it is no longer necessary to enrol again or retrain the system from scratch in order to cope with the changing environment. This convenience can significantly reduce the cost of maintaining a biometric system. Despite these advantages, there are several open issues involved with these systems. For mis-classification error (false acceptance) by the biometric system, cause adaptation using impostor sample. However, continuous research efforts are directed to resolve the open issues associated to the field of adaptive biometrics. More information about adaptive biometric systems can be found in the critical review by Rattani et al.

Recent advances in emerging biometrics

In recent times, biometrics based on brain (electroencephalogram) and heart (electrocardiogram) signals have emerged. The research group at University of Kent led by Ramaswamy Palaniappan has shown that people have certain distinct brain and heart patterns that are specific for each individual. Another example is finger vein recognition, using pattern-recognition techniques, based on images of human vascular patterns. The advantage of such 'futuristic' technology is that it is more fraud resistant compared to conventional biometrics like fingerprints. However, such technology is generally more cumbersome and still has issues such as lower accuracy and poor reproducibility over time. This new generation of biometrical systems is called biometrics of intent and it aims to scan intent. The technology will analyze physiological features such as eye movement, body temperature, breathing etc. and predict dangerous behaviour or hostile intent before it materializes into action.

On the portability side of biometric products, more and more vendors are embracing significantly miniaturized biometric authentication systems (BAS) thereby driving elaborate cost savings, especially for large-scale deployments.

Operator signatures

An operator signature is a biometric mode where the manner in which a person using a device or complex system is recorded as a verification template. One potential use for this type of biometric signature is to distinguish among remote users of telerobotic surgery systems that utilize public networks for communication.

Proposed requirement for certain public networks

John Michael (Mike) McConnell, a former vice admiral in the United States Navy, a former Director of U.S. National Intelligence, and Senior Vice President of Booz Allen Hamilton promoted the development of a future capability to require biometric authentication to access certain public networks in his keynote speech at the 2009 Biometric Consortium Conference.

A basic premise in the above proposal is that the person that has uniquely authenticated themselves using biometrics with the computer is in fact also the agent performing potentially malicious actions from that computer. However, if control of the computer has been subverted, for example in which the computer is part of a botnet controlled by a hacker, then knowledge of the identity of the user at the terminal does not materially improve network security or aid law enforcement activities.

Recently, another approach to biometric security was developed, this method scans the entire body of prospects to guarantee a better identification of this prospect. This method is not globally accepted because it is very complex and prospects are concerned about their privacy.

Animal biometrics

Rather than tags or tattoos, biometric techniques may be used to identify individual animals: zebra stripes, blood vessel patterns in rodent ears, muzzle prints, bat wing patterns, primate facial recognition and koala spots have all been tried.

Video

Videos have become a pronounced way of identifying information. There are features in videos that look at how intense certain parts of a frame are compared to others which help with identification.

Issues and concerns

Surveillance humanitarianism in times of crisis

Biometrics are employed by many aid programs in times of crisis in order to prevent fraud and ensure that resources are properly available to those in need. Humanitarian efforts are motivated by promoting the welfare of individuals in need, however the use of biometrics as a form of surveillance humanitarianism can create conflict due to varying interests of the groups involved in the particular situation. Disputes over the use of biometrics between aid programs and party officials stalls the distribution of resources to people that need help the most. In July of 2019, the United Nations World Food Program and Houthi Rebels were involved in a large dispute over the use of biometrics to ensure resources are provided to the hundreds of thousands of civilians in Yemen whose lives are threatened. The refusal to cooperate with the interests of the United Nations World Food Program resulted in the suspension of food aid to the Yemen population. The use of biometrics may provide aid programs with valuable information, however its potential solutions may not be best suited for chaotic times of crisis. Conflicts that are caused by deep-rooted political problems, in which the implementation of biometrics may not provide a long-term solution.

Human dignity

Biometrics have been considered also instrumental to the development of state authority (to put it in Foucauldian terms, of discipline and biopower). By turning the human subject into a collection of biometric parameters, biometrics would dehumanize the person, infringe bodily integrity, and, ultimately, offend human dignity.

In a well-known case, Italian philosopher Giorgio Agamben refused to enter the United States in protest at the United States Visitor and Immigrant Status Indicator (US-VISIT) program's requirement for visitors to be fingerprinted and photographed. Agamben argued that gathering of biometric data is a form of bio-political tattooing, akin to the tattooing of Jews during the Holocaust. According to Agamben, biometrics turn the human persona into a bare body. Agamben refers to the two words used by Ancient Greeks for indicating "life", zoe, which is the life common to animals and humans, just life; and bios, which is life in the human context, with meanings and purposes. Agamben envisages the reduction to bare bodies for the whole humanity. For him, a new bio-political relationship between citizens and the state is turning citizens into pure biological life (zoe) depriving them from their humanity (bios); and biometrics would herald this new world.

In Dark Matters: On the Surveillance of Blackness, surveillance scholar Simone Browne formulates a similar critique as Agamben, citing a recent study relating to biometrics R&D that found that the gender classification system being researched "is inclined to classify Africans as males and Mongoloids as females." Consequently, Browne argues that the conception of an objective biometric technology is difficult if such systems are subjectively designed, and are vulnerable to cause errors as described in the study above. The stark expansion of biometric technologies in both the public and private sector magnifies this concern. The increasing commodification of biometrics by the private sector adds to this danger of loss of human value. Indeed, corporations value the biometric characteristics more than the individuals value them. Browne goes on to suggest that modern society should incorporate a "biometric consciousness" that "entails informed public debate around these technologies and their application, and accountability by the state and the private sector, where the ownership of and access to one's own body data and other intellectual property that is generated from one's body data must be understood as a right."

Other scholars have emphasized, however, that the globalized world is confronted with a huge mass of people with weak or absent civil identities. Most developing countries have weak and unreliable documents and the poorer people in these countries do not have even those unreliable documents. Without certified personal identities, there is no certainty of right, no civil liberty. One can claim her rights, including the right to refuse to be identified, only if she is an identifiable subject, if she has a public identity. In such a sense, biometrics could play a pivotal role in supporting and promoting respect for human dignity and fundamental rights.

The biometrics of intent poses further risks. In his paper in Harvard International Review, Prof Nayef Al-Rodhan cautions about the high risks of miscalculations, wrongful accusations and infringements of civil liberties. Critics in the US have also signalled a conflict with the 4th Amendment.

Privacy and discrimination

It is possible that data obtained during biometric enrollment may be used in ways for which the enrolled individual has not consented. For example, most biometric features could disclose physiological and/or pathological medical conditions (e.g., some fingerprint patterns are related to chromosomal diseases, iris patterns could reveal genetic sex, hand vein patterns could reveal vascular diseases, most behavioral biometrics could reveal neurological diseases, etc.). Moreover, second generation biometrics, notably behavioral and electro-physiologic biometrics (e.g., based on electrocardiography, electroencephalography, electromyography), could be also used for emotion detection.

There are three categories of privacy concerns:

1. Unintended functional scope: The authentication goes further than authentication, such as finding a tumor.
2. Unintended application scope: The authentication process correctly identifies the subject when the subject did not wish to be identified.
3. Covert identification: The subject is identified without seeking identification or authentication, i.e. a subject's face is identified in a crowd.

Danger to owners of secured items

When thieves cannot get access to secure properties, there is a chance that the thieves will stalk and assault the property owner to gain access. If the item is secured with a biometric device, the damage to the owner could be irreversible, and potentially cost more than the secured property. For example, in 2005, Malaysian car thieves cut off the finger of a Mercedes-Benz S-Class owner when attempting to steal the car.

Presentation attacks

In the context of biometric systems, presentation attacks may also be called "spoofing attacks".

As per the recent ISO/IEC 30107 standard, presentation attacks are defined as "presentation to the biometric capture subsystem with the goal of interfering with the operation of the biometric system". These attacks can be either impersonation or obfuscation attacks. Impersonation attacks try to gain access by pretending to be someone else. Obfuscation attacks may, for example, try to evade face detection and face recognition systems.

Recently several methods have been proposed to counteract presentation attacks. Today's sophisticated biometric systems use "liveness" elements to detect spoofs (a.k.a. fake images), and some fingerprint scanners have pulse detectors. Automated detection of a presentation attack is called a "presentation attack detection" (PAD).

Cancelable biometrics

One advantage of passwords over biometrics is that they can be re-issued. If a token or a password is lost or stolen, it can be cancelled and replaced by a newer version. This is not naturally available in biometrics. If someone's face is compromised from a database, they cannot cancel or reissue it. If the electronic biometric identifier is stolen, it is nearly impossible to change a biometric feature. This renders the person's biometric feature questionable for future use in authentication, such as the case with the hacking of security-clearance-related background information from the Office of Personnel Management (OPM) in the United States.

Cancelable biometrics is a way in which to incorporate protection and the replacement features into biometrics to create a more secure system. It was first proposed by Ratha et al. 

"Cancelable biometrics refers to the intentional and systematically repeatable distortion of biometric features in order to protect sensitive user-specific data. If a cancelable feature is compromised, the distortion characteristics are changed, and the same biometrics is mapped to a new template, which is used subsequently. Cancelable biometrics is one of the major categories for biometric template protection purpose besides biometric cryptosystem." In biometric cryptosystem, "the error-correcting coding techniques are employed to handle intraclass variations." This ensures a high level of security but has limitations such as specific input format of only small intraclass variations.

Several methods for generating new exclusive biometrics have been proposed. The first fingerprint-based cancelable biometric system was designed and developed by Tulyakov et al. Essentially, cancelable biometrics perform a distortion of the biometric image or features before matching. The variability in the distortion parameters provides the cancelable nature of the scheme. Some of the proposed techniques operate using their own recognition engines, such as Teoh et al. and Savvides et al., whereas other methods, such as Dabbah et al., take the advantage of the advancement of the well-established biometric research for their recognition front-end to conduct recognition. Although this increases the restrictions on the protection system, it makes the cancellable templates more accessible for available biometric technologies

Soft biometrics

Soft biometrics traits are physical, behavioral or adhered human characteristics that have been derived from the way human beings normally distinguish their peers (e.g. height, gender, hair color). They are used to complement the identity information provided by the primary biometric identifiers. Although soft biometric characteristics lack the distinctiveness and permanence to recognize an individual uniquely and reliably, and can be easily faked, they provide some evidence about the users identity that could be beneficial. In other words, despite the fact they are unable to individualize a subject, they are effective in distinguishing between people. Combinations of personal attributes like gender, race, eye color, height and other visible identification marks can be used to improve the performance of traditional biometric systems. Most soft biometrics can be easily collected and are actually collected during enrollment. Two main ethical issues are raised by soft biometrics. First, some of soft biometric traits are strongly cultural based; e.g., skin colors for determining ethnicity risk to support racist approaches, biometric sex recognition at the best recognizes gender from tertiary sexual characters, being unable to determine genetic and chromosomal sexes; soft biometrics for aging recognition are often deeply influenced by ageist stereotypes, etc. Second, soft biometrics have strong potential for categorizing and profiling people, so risking of supporting processes of stigmatization and exclusion.

International sharing of biometric data

Many countries, including the United States, are planning to share biometric data with other nations.

In testimony before the US House Appropriations Committee, Subcommittee on Homeland Security on "biometric identification" in 2009, Kathleen Kraninger and Robert A Mocny commented on international cooperation and collaboration with respect to biometric data, as follows:

To ensure we can shut down terrorist networks before they ever get to the United States, we must also take the lead in driving international biometric standards. By developing compatible systems, we will be able to securely share terrorist information internationally to bolster our defenses. Just as we are improving the way we collaborate within the U.S. Government to identify and weed out terrorists and other dangerous people, we have the same obligation to work with our partners abroad to prevent terrorists from making any move undetected. Biometrics provide a new way to bring terrorists' true identities to light, stripping them of their greatest advantage—remaining unknown.

According to an article written in 2009 by S. Magnuson in the National Defense Magazine entitled "Defense Department Under Pressure to Share Biometric Data" the United States has bilateral agreements with other nations aimed at sharing biometric data. To quote that article:

Miller [a consultant to the Office of Homeland Defense and America's security affairs] said the United States has bilateral agreements to share biometric data with about 25 countries. Every time a foreign leader has visited Washington during the last few years, the State Department has made sure they sign such an agreement.

Likelihood of full governmental disclosure

Certain members of the civilian community are worried about how biometric data is used but full disclosure may not be forthcoming. In particular, the Unclassified Report of the United States' Defense Science Board Task Force on Defense Biometrics states that it is wise to protect, and sometimes even to disguise, the true and total extent of national capabilities in areas related directly to the conduct of security-related activities. This also potentially applies to Biometrics. It goes on to say that this is a classic feature of intelligence and military operations. In short, the goal is to preserve the security of 'sources and methods'.

Countries applying biometrics

Countries using biometrics include Australia, Brazil, Canada, Cyprus, Greece, China, Gambia, Germany, India, Iraq, Ireland, Israel, Italy, Malaysia, Netherlands, New Zealand, Nigeria, Norway, Pakistan, South Africa, Saudi Arabia, Tanzania, Ukraine, United Arab Emirates, United Kingdom, United States and Venezuela.

Among low to middle income countries, roughly 1.2 billion people have already received identification through a biometric identification program.

There are also numerous countries applying biometrics for voter registration and similar electoral purposes. According to the International IDEA's ICTs in Elections Database, some of the countries using (2017) Biometric Voter Registration (BVR) are Armenia, Angola, Bangladesh, Bhutan, Bolivia, Brazil, Burkina Faso, Cambodia, Cameroon, Chad, Colombia, Comoros, Congo (Democratic Republic of), Costa Rica, Ivory Coast, Dominican Republic, Fiji, Gambia, Ghana, Guatemala, India, Iraq, Kenya, Lesotho, Liberia, Malawi, Mali, Mauritania, Mexico, Morocco, Mozambique, Namibia, Nepal, Nicaragua, Nigeria, Panama, Peru, The Philippines, Senegal, Sierra Leone, Solomon Islands, Somaliland, Swaziland, Tanzania, Uganda, Uruguay, Venezuela, Yemen, Zambia, and Zimbabwe.

India's national ID program

India's national ID program called Aadhaar is the largest biometric database in the world. It is a biometrics-based digital identity assigned for a person's lifetime, verifiable online instantly in the public domain, at any time, from anywhere, in a paperless way. It is designed to enable government agencies to deliver a retail public service, securely based on biometric data (fingerprint, iris scan and face photo), along with demographic data (name, age, gender, address, parent/spouse name, mobile phone number) of a person. The data is transmitted in encrypted form over the internet for authentication, aiming to free it from the limitations of physical presence of a person at a given place.

About 550 million residents have been enrolled and assigned 480 million Aadhaar national identification numbers as of 7 November 2013. It aims to cover the entire population of 1.2 billion in a few years. However, it is being challenged by critics over privacy concerns and possible transformation of the state into a surveillance state, or into a Banana republic.§ The project was also met with mistrust regarding the safety of the social protection infrastructures. To tackle the fear amongst the people, India's supreme court put a new ruling into action that stated that privacy from then on was seen as a fundamental right. On 24 August 2017 this new law was established.