JPWO2020123773A5

JPWO2020123773A5 -

Info

Publication number: JPWO2020123773A5
Application number: JP2021533524A
Authority: JP
Publication date: 2022-12-20

Description

ＯＲＦ１のＮ２２ドメイン中のＹＮＰＸ^２ＤＸＧＸ^２Ｎモチーフ（配列番号８２９）の二次構造も、それを取り囲む保存二次構造を有する。５～６ＡＡβ鎖で開始して、これは、モチーフの１位のチロシン（Ｙ）後に切断し、モチーフのほとんどは、８～９ＡＡランダムコイルとして末端アスパラギン（Ｎ）まで配列し、その地点で、７～８ＡＡの別のβ鎖が開始する。例示的なアネロウイルス（Ａｎｅｌｌｏｖｉｒｕｓ）ＯＲＦ１Ｎ２２モチーフ配列のアラインメントを図４８に示す。このモチーフ中のチロシンはβ鎖を切断し、第２β鎖が、モチーフの末端アスパラギンで開始する。
特定の実施形態では、例えば、以下が提供される：
(項目１)
以下：
（Ｉ）以下：
（ａ）プロモータエレメントと、
（ｂ）外性エフェクターをコード化する核酸配列であって、前記核酸配列が、前記プロモータエレメントに作動可能に連結されており、前記外性エフェクターが、成長ホルモン（例えば、ｈＧＨ）である、核酸配列と；
（ｃ）以下：
配列番号５４のヌクレオチド３２３～３９３の核酸配列、若しくはそれに対して少なくとも９０％同一の核酸配列を含む５’ＵＴＲドメインと、
を含む遺伝子エレメント；
（ＩＩ）アネロウイルス（Ａｎｅｌｌｏｖｉｒｕｓ）ＯＲＦ１核酸によりコード化されるポリペプチド、例えばＯＲＦ１分子を含むタンパク質性外層
を含む合成アネロソームにおいて；
前記遺伝子エレメントが、前記タンパク質性外層内に閉じ込められており；且つ
前記合成アネロソームが、前記遺伝子エレメントをヒト細胞内に送達することができる、合成アネロソーム。
(項目２)
前記ＯＲＦ１分子が、配列番号２１７のアミノ酸配列、又はそれに対して少なくとも９０％の同一性を有するアミノ酸配列を含む、項目１に記載の合成アネロソーム。
(項目３)
前記ＯＲＦ１分子が、配列番号５４のヌクレオチド６１２～２６１２によりコード化される、項目１～２のいずれか１項に記載の合成アネロソーム。
(項目４)
前記遺伝子エレメントが、配列番号５４のヌクレオチド２８６８～２９２９の核酸配列、又はそれに対して少なくとも８５％の配列同一性を有する核酸配列を含む、項目１～３のいずれか１項に記載の合成アネロソーム。
(項目５)
前記ＯＲＦ１分子が、表１６に列挙されるａｒｇリッチ領域、ゼリーロールドメイン、超可変ドメイン、Ｎ２２ドメイン、及び／又はＣ末端ドメインのアミノ酸配列、又はそれに対して少なくとも８５％の同一性を有するアミノ酸配列のうち１つ若しくは複数を含むアミノ酸配列を含む、項目１～４のいずれか１項に記載の合成アネロソーム。
(項目６)
前記ＯＲＦ１分子が、配列番号５８のアミノ酸配列、又はそれに対して少なくとも８５％の配列同一性を有する核酸配列を含む、項目１～５のいずれか１項に記載の合成アネロソーム。
(項目７)
表１６に列挙されるＯＲＦ２、ＯＲＦ２／２、ＯＲＦ２／３、ＯＲＦ１／１、若しくはＯＲＦ１／２のアミノ酸配列、又はそれに対して少なくとも８５％の同一性を有するアミノ酸配列を含むポリペプチドをさらに含む、項目１～６のいずれか１項に記載の合成アネロソーム。
(項目８)
前記遺伝子エレメントが、表１６に列挙されるＯＲＦ１、ＯＲＦ２、ＯＲＦ２／２、ＯＲＦ２／３、ＯＲＦ１／１、若しくはＯＲＦ１／２のアミノ酸配列、又はそれに対して少なくとも８５％の同一性を有するアミノ酸配列をコード化する、項目１～７のいずれか１項に記載の合成アネロソーム。
(項目９)
前記合成アネロソームが、表１６に列挙されるＯＲＦ２、ＯＲＦ２／２、ＯＲＦ２／３、ＴＡＩＰ、ＯＲＦ１／１、若しくはＯＲＦ１／２のアミノ酸配列、又はそれに対して少なくとも８５％の同一性を有するアミノ酸配列を含むポリペプチドを含まない、項目１～８のいずれか１項に記載の合成アネロソーム。
(項目１０)
前記遺伝子エレメントが、表１６に列挙されるＯＲＦ１、ＯＲＦ２、ＯＲＦ２／２、ＯＲＦ２／３、ＯＲＦ１／１、若しくはＯＲＦ１／２のアミノ酸配列、又はそれに対して少なくとも８５％の同一性を有するアミノ酸配列をコード化しない、項目１～９のいずれか１項に記載の合成アネロソーム。
(項目１１)
前記ＯＲＦ１分子が、アミノ酸配列ＹＮＰＸ ^２ＤＸＧＸ ^２Ｎ（配列番号８２９）（Ｘ ^ｎは、各々独立して、任意のｎ個のアミノ酸の連続した配列である）を含む、項目１～１０のいずれか１項に記載の合成アネロソーム。
(項目１２)
前記ＯＲＦ１分子が、アミノ酸配列ＹＮＰＸ ^２ＤＸＧＸ ^２Ｎ（配列番号８２９）とフランキングする第１β鎖及び第２β鎖をさらに含み、例えば、前記第１β鎖は、前記アミノ酸配列ＹＮＰＸ ^２ＤＸＧＸ ^２Ｎ（配列番号８２９）のチロシン（Ｙ）残基を含み、且つ／又は前記第２β鎖は、前記アミノ酸配列ＹＮＰＸ ^２ＤＸＧＸ ^２Ｎ（配列番号８２９）の第２アスパラギン（Ｎ）残基（ＮからＣ）を含む、項目１１に記載の合成アネロソーム。
(項目１３)
前記ＯＲＦ１分子が、Ｎ末端からＣ末端方向の順で、第１β鎖、第２β鎖、第１αヘリックス、第３β鎖、第４β鎖、第５β鎖、第２αヘリックス、第６β鎖、第７β鎖、第８β鎖、及び第９β鎖を含む、項目１～１２のいずれか１項に記載の合成アネロソーム。
(項目１４)
前記遺伝子エレメントが、宿主細胞内でのローリングサークル複製により増幅されて、例えば、少なくとも８コピーを産生することができる、項目１～１３のいずれか１項に記載の合成アネロソーム。
(項目１５)
前記遺伝子エレメントが、一本鎖である、項目１～１４のいずれか１項に記載の合成アネロソーム。
(項目１６)
前記遺伝子エレメントが、環状である、項目１～１５のいずれか１項に記載の合成アネロソーム。
(項目１７)
前記遺伝子エレメントが、ＤＮＡである、項目１～１６のいずれか１項に記載の合成アネロソーム。
(項目１８)
前記遺伝子エレメントが、マイナス鎖ＤＮＡである、項目１～１７のいずれか１項に記載の合成アネロソーム。
(項目１９)
前記遺伝子エレメントが、前記細胞に進入する前記アネロソームの１０％、８％、６％、４％、３％、２％、１％、０．５％、０．２％、０．１％未満の頻度で組み込まれ、例えば、前記合成アネロソームは非統合性である、項目１～１８のいずれか１項に記載の合成アネロソーム。
(項目２０)
前記遺伝子エレメントが、表１６－１に示すコンセンサス５’ＵＴＲ核酸配列の配列を含む、項目１～１９のいずれか１項に記載の合成アネロソーム。
(項目２１)
前記遺伝子エレメントが、表１６－２に示すコンセンサスＧＣリッチ領域の配列を含む、項目１～２０のいずれか１項に記載の合成アネロソーム。
(項目２２)
前記遺伝子エレメントが、少なくとも１００ヌクレオチド長の配列を含み、これは、少なくとも７０％（例えば、約７０～１００％、７５～９５％、８０～９５％、８５～９５％、若しくは８５～９０％）の位置のＧ又はＣから構成される、項目１～２１のいずれか１項に記載の合成アネロソーム。
(項目２３)
前記遺伝子エレメントが、配列番号１２０の核酸配列を含む、項目１～２２のいずれか１項に記載の合成アネロソーム。
(項目２４)
前記プロモータエレメントが、野生型アネロウイルス（Ａｎｅｌｌｏｖｉｒｕｓ）に対して外性である、項目１～２３のいずれか１項に記載の合成アネロソーム。
(項目２５)
前記プロモータエレメントが、野生型アネロウイルス（Ａｎｅｌｌｏｖｉｒｕｓ）に対して内在性である、項目１～２４のいずれか１項に記載の合成アネロソーム。
(項目２６)
前記遺伝子エレメントが、約１．５～２．０、２．０～２．５、２．５～３．０、３．０～３．５、３．１～３．６、３．２～３．７、３．３～３．８、３．４～３．９、３．５～４．０、４．０～４．５、又は４．５～５．０ｋｂの長さを有する、項目１～２５のいずれか１項に記載の合成アネロソーム。
(項目２７)
前記合成アネロソームが、例えば、インビトロで、ヒト細胞、例えば、血液細胞、皮膚細胞、筋肉細胞、神経細胞、脂肪細胞、内皮細胞、免疫細胞、肝細胞、肺上皮細胞に感染することができる、項目１～２６のいずれか１項に記載の合成アネロソーム。
(項目２８)
実質的に非免疫原性であり、例えば、実施例４に記載の方法に従って検出される場合、例えば、検出可能な及び／又は不要な免疫応答を誘導しない、項目１～２７のいずれか１項に記載の合成アネロソーム。
(項目２９)
前記実質的に非免疫原性のアネロソームが、被験者において、免疫応答が欠損する対照被験者における効力の少なくとも約１０％、２０％、３０％、４０％、５０％、６０％、７０％、８０％、９０％、９５％、又は１００％である効力を有する、項目２８に記載の合成アネロソーム。
(項目３０)
少なくとも１０００の前記アネロソームの集団が、少なくとも約１００コピー（例えば、少なくとも１、２、３、４、５、１０、２０、３０、４０、５０、１００、２００、３００、４００、５００、６００、７００、８００、９００、又は１０００コピー）の遺伝子エレメントを１つ若しくは複数のヒト細胞内に送達することができる、項目１～２９のいずれか１項に記載の合成アネロソーム。
(項目３１)
以下：
（Ｉ）以下：
（ａ）プロモータエレメントと、
（ｂ）外性エフェクターをコード化する核酸配列であって、前記核酸配列は、前記プロモータエレメントに作動可能に連結されており、
前記外性エフェクターが、以下：
（ｉ）増殖因子若しくは増殖因子受容体、又はサイトカイン若しくはサイトカイン受容体に結合する抗体分子；
（ｉｉ）酵素若しくはその機能性変異体；
（ｉｉｉ）ホルモン若しくはその機能性変異体；
（ｉｖ）サイトカイン若しくはその機能性変異体）；
（ｖ）補体阻害剤；
（ｖｉ）増殖因子、
（ｖｉ）増殖因子阻害剤、
（ｖｉｉ）凝固因子、又は
（ｖｉｉｉ）ＳＴＩＮＧ／ｃＧＡＳシグナル伝達のモジュレータ
から選択される分泌治療薬である、核酸配列と、
（ｃ）以下：
配列番号５４のヌクレオチド３２３～３９３の核酸配列、若しくはそれに対して少なくとも８５％同一の核酸配列を含む５’ＵＴＲドメインと、
を含む遺伝子エレメント；
（ＩＩ）ＯＲＦ１核酸によりコード化されるポリペプチド、例えば、ＯＲＦ１分子を含むタンパク質性外層
を含む合成アネロソームにおいて、
前記遺伝子エレメントが、前記タンパク質性外層内に閉じ込められており；且つ
前記合成アネロソームが、前記遺伝子エレメントをヒト細胞に送達することができる、合成アネロソーム。
(項目３２)
項目１～３１のいずれか１項に記載の合成アネロソーム、及び薬学的に許容される担体又は賦形剤を含む、医薬組成物。
(項目３３)
少なくとも１０ ^３、１０ ^４、１０５、１０ ^６、１０ ^７、１０ ^８、又は１０ ^９の合成アネロソームを含む、項目３２に記載の医薬組成物。
(項目３４)
前記医薬組成物が、予め定めた比の粒子：感染性単位（例えば、＜３００：１、＜２００：１、＜１００：１、若しくは＜５０：１）を有する、項目３２又は３３に記載の医薬組成物。
(項目３５)
以下：
（ｉ）項目１～３４のいずれか１項に記載の合成アネロソームの前記遺伝子エレメントの配列を含む第１核酸（例えば、二本鎖又は一本鎖環状ＤＮＡ）、及び
（ｉｉ）例えば、表１６に列挙されるＯＲＦ１、ＯＲＦ２、ＯＲＦ２／２、ＯＲＦ２／３、ＯＲＦ１／１、若しくはＯＲＦ１／２から選択されるアミノ酸配列、又はそれに対して少なくとも８５％の配列同一性を有するアミノ酸配列の１つ若しくは複数をコード化する第２核酸配列
を含む、反応混合物。
(項目３６)
前記第１核酸及び第２核酸が、同じ核酸分子中にある、項目３５に記載の反応混合物。
(項目３７)
前記第１核酸及び第２核酸が、異なる核酸分子である、項目３５に記載の反応混合物。
(項目３８)
前記第１核酸及び第２核酸が、異なる核酸分子であり、且つ、前記第２核酸が、二本鎖環状ＤＮＡとして提供される、項目３５に記載の反応混合物。
(項目３９)
前記第１核酸及び第２核酸が、異なる核酸分子であり、且つ、前記第１及び第２核酸が、二本鎖環状ＤＮＡとして提供される、項目３５に記載の反応混合物。
(項目４０)
前記第２核酸配列が、ヘルパー細胞又はヘルパーウイルスに含まれる、項目３７に記載の反応混合物。
(項目４１)
合成アネロソームを作製する方法であって、前記方法が、以下：
ａ）以下：
（ｉ）項目１～４１のいずれか１項に記載の合成アネロソームの遺伝子エレメントの前記核酸配列を含む第１核酸分子と、
（ｉｉ）例えば、表１６のいずれかに列挙される、ＯＲＦ１、ＯＲＦ２、ＯＲＦ２／２、ＯＲＦ２／３、ＯＲＦ１／１、若しくはＯＲＦ１／２から選択されるアミノ酸配列、又はそれに対して少なくとも８５％の配列同一性を有するアミノ酸配列の１つ若しくは複数をコード化する第２核酸分子と、
を含む宿主細胞を用意するステップ；及び
ｂ）合成アネロソームを作製するのに好適な条件下で前記宿主細胞をインキュベートするステップを含み、
それによって前記合成アネロソームを作製する方法。
(項目４２)
ステップ（ａ）の前に、前記第１核酸分子及び／又は前記第２核酸分子を前記細胞に導入するステップをさらに含む、項目４１に記載の方法。
(項目４３)
前記第２核酸分子が、前記第１核酸分子の前に、それと同時に、又はその後に前記宿主細胞に導入される、項目４２に記載の方法。
(項目４４)
前記第２核酸分子が、前記宿主細胞のゲノム中に組み込まれる、項目４１又は４２のいずれか１項に記載の方法。
(項目４５)
前記第２核酸分子が、ヘルパー（例えば、ヘルパープラスミド又はヘルパーウイルスのゲノム）である、項目４１～４４のいずれか１項に記載の方法。
(項目４６)
第２核酸分子が、アミノ酸配列［Ｗ／Ｆ］Ｘ ^７ＨＸ ^３ＣＸ ^１ＣＸ ^５Ｈ（配列番号９４９）（Ｘ ^ｎは、任意のｎ個のアミノ酸の連続した配列である）を含むＯＲＦ２分子をコード化する、項目４１～４４のいずれか１項に記載の方法。
(項目４７)
合成アネロソーム調製物を製造する方法であって、前記方法が、以下：
ａ）項目１～３１のいずれか１項に記載の複数の合成アネロソーム、項目３２～３４のいずれか１項に記載の医薬組成物、又は項目３５～４０のいずれか１項に記載の反応混合物を用意するステップ；
ｂ）任意選択で、以下：本明細書に記載の混入物、光学密度測定値（例えば、ＯＤ２６０）、粒子数（例えば、ＨＰＬＣにより）、感染力（例えば、粒子：感染単位比）のうち１つ又は複数について前記複数を評価するステップ；並びに
ｃ）例えば、（ｂ）のパラメータの１つ又は複数が、指定閾値を満たす場合、例えば、被験者への投与に好適な医薬組成物として、前記複数の合成アネロソームを製剤化するステップ
を含む、方法。
(項目４８)
以下：
（ｉ）項目１～４７のいずれか１項に記載の合成アネロソームの遺伝子エレメントの前記核酸配列を含む第１核酸分子、及び
（ｉｉ）任意選択で、表１６のいずれかに列挙されるＯＲＦ１、ＯＲＦ２、ＯＲＦ２／２、ＯＲＦ２／３、ＯＲＦ１／１、若しくはＯＲＦ１／２から選択されるアミノ酸配列、又はそれに対して少なくとも８５％の配列同一性を有するアミノ酸配列の１つ若しくは複数をコード化する第２核酸分子；
を含む宿主細胞。
(項目４９)
外性エフェクター（例えば、治療用外性エフェクター）を哺乳動物細胞に送達する方法において、以下：
（ａ）項目１～４８のいずれか１項に記載の合成アネロソームを用意するステップ；及び
（ｂ）哺乳動物細胞を前記合成アネロソームと接触させるステップ
を含み；
ここで、前記合成アネロソームは、前記遺伝子エレメントを前記哺乳動物細胞に送達することができ；
任意選択で、前記遺伝子エレメントを宿主細胞中の前記タンパク質性外層内に閉じ込めるのに好適な条件下で、前記遺伝子エレメントを前記宿主細胞に導入することにより、前記合成アネロソームが生産され；
これによって、前記治療用外性エフェクターを前記哺乳動物細胞に送達する、方法。
(項目５０)
前記遺伝子エレメントの宿主細胞への送達を目的とする、項目１～３１のいずれか１項に記載の合成アネロソーム又は項目３２～３４のいずれか１項に記載の医薬組成物の使用。
(項目５１)
被験者の疾患又は障害の治療を目的とする、項目１～３１のいずれか１項に記載の合成アネロソーム又は項目３２～３４のいずれか１項に記載の医薬組成物の使用。
(項目５２)
被験者の疾患又は障害の治療に使用するための、項目１～３１のいずれか１項に記載の合成アネロソーム又は項目３２～３４のいずれか１項に記載の医薬組成物。
(項目５３)
被験者の疾患又は障害を治療する方法であって、前記方法が、項目１～３１のいずれか１項に記載の合成アネロソーム又は項目３２～３４のいずれか１項に記載の医薬組成物を前記被験者に投与するステップを含む、方法。
(項目５４)
被験者の疾患又は障害の治療のための薬剤の製造における項目１～３１のいずれか１項に記載の合成アネロソーム又は項目３２～３４のいずれか１項に記載の医薬組成物の使用。 The ^secondary structure of the ^YNPX2DXGX2N motif (SEQ ID NO:829) in the N22 domain of ORF1 also has conserved secondary structure surrounding it. Starting with the 5-6 AA β-strand, this cleaves after the tyrosine (Y) at position 1 of the motif and most of the motif is arranged as 8-9 AA random coils to the terminal asparagine (N), at which point the 7 Another β-strand of ~8AA starts. An alignment of exemplary Anellovirus ORF1 N22 motif sequences is shown in FIG. A tyrosine in this motif cleaves the β-strand and the second β-strand begins at the terminal asparagine of the motif.
In certain embodiments, for example, the following are provided:
(Item 1)
Less than:
(I) Below:
(a) a promoter element;
(b) a nucleic acid sequence encoding an exogenous effector, wherein said nucleic acid sequence is operably linked to said promoter element and said exogenous effector is a growth hormone (e.g., hGH); an array;
(c) below:
a 5'UTR domain comprising the nucleic acid sequence of nucleotides 323-393 of SEQ ID NO:54, or a nucleic acid sequence at least 90% identical thereto;
a genetic element comprising;
(II) A proteinaceous outer layer comprising a polypeptide encoded by an Anellovirus ORF1 nucleic acid, such as an ORF1 molecule
in a synthetic anerosome comprising;
said genetic element is enclosed within said proteinaceous outer layer; and
A synthetic anerosome, wherein said synthetic anerosome is capable of delivering said genetic element into a human cell.
(Item 2)
The synthetic anellosome of item 1, wherein said ORF1 molecule comprises the amino acid sequence of SEQ ID NO: 217 or an amino acid sequence having at least 90% identity thereto.
(Item 3)
3. The synthetic anellosome of any one of items 1-2, wherein said ORF1 molecule is encoded by nucleotides 612-2612 of SEQ ID NO:54.
(Item 4)
4. The synthetic anellosome of any one of items 1-3, wherein said genetic element comprises the nucleic acid sequence of nucleotides 2868-2929 of SEQ ID NO: 54, or a nucleic acid sequence having at least 85% sequence identity thereto.
(Item 5)
said ORF1 molecule is an arg-rich region, jelly roll domain, hypervariable domain, N22 domain, and/or C-terminal domain amino acid sequence listed in Table 16, or an amino acid sequence having at least 85% identity thereto 5. The synthetic anelosome of any one of items 1-4, comprising an amino acid sequence comprising one or more of:
(Item 6)
Synthetic anellosome according to any one of items 1 to 5, wherein said ORF1 molecule comprises the amino acid sequence of SEQ ID NO: 58 or a nucleic acid sequence having at least 85% sequence identity thereto.
(Item 7)
further comprising a polypeptide comprising an amino acid sequence of ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 listed in Table 16, or an amino acid sequence having at least 85% identity thereto; A synthetic anerosome according to any one of items 1-6.
(Item 8)
wherein said genetic element comprises an amino acid sequence of ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 listed in Table 16, or an amino acid sequence having at least 85% identity thereto; A synthetic anelosome according to any one of items 1 to 7, which encodes.
(Item 9)
wherein said synthetic anerosomes have an amino acid sequence of ORF2, ORF2/2, ORF2/3, TAIP, ORF1/1, or ORF1/2 listed in Table 16, or an amino acid sequence having at least 85% identity thereto; 9. The synthetic anelosome of any one of items 1-8, free of the containing polypeptide.
(Item 10)
wherein said genetic element comprises an amino acid sequence of ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 listed in Table 16, or an amino acid sequence having at least 85% identity thereto; A synthetic anelosome according to any one of items 1 to 9, which is not encoded.
(Item 11)
any of items 1-10, wherein said ORF1 molecule comprises the amino acid sequence YNPX ² DXGX ² N (SEQ ID NO: 829) (each X ⁿ is independently any consecutive sequence of n amino acids) 2. The synthetic anellosome according to item 1.
(Item 12)
Said ORF1 molecule further comprises first and second β-strands flanking the amino acid sequence YNPX ² DXGX ² N (SEQ ID NO: 829), for example, said first β-strand comprises said amino acid sequence YNPX ² DXGX ² N (sequence 829), and/or said second β-strand comprises a second asparagine (N) residue (N to C) of said amino acid sequence YNPX ² DXGX ² N (SEQ ID NO: 829). 12. The synthetic anellosome of item 11, comprising:
(Item 13)
The ORF1 molecule is, in order from the N-terminus to the C-terminus, 1st β-strand, 2nd β-strand, 1st α-helix, 3rd β-strand, 4th β-strand, 5th β-strand, 2nd α-helix, 6th β-strand, 7th β-strand , β-strand 8, and β-strand 9, according to any one of items 1 to 12.
(Item 14)
14. A synthetic anellosome according to any one of items 1 to 13, wherein said genetic element is capable of being amplified by rolling circle replication in a host cell to produce, for example, at least 8 copies.
(Item 15)
15. The synthetic anelosome of any one of items 1-14, wherein said genetic element is single-stranded.
(Item 16)
16. The synthetic anelosome of any one of items 1-15, wherein said genetic element is circular.
(Item 17)
17. The synthetic anelosome of any one of items 1-16, wherein said genetic element is DNA.
(Item 18)
18. The synthetic anelosome of any one of items 1-17, wherein said genetic element is minus-strand DNA.
(Item 19)
said genetic element is less than 10%, 8%, 6%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1% of said anellosomes entering said cell Synthetic anerosome according to any one of items 1 to 18, which is integrated at a frequency, eg said synthetic anerosome is non-integrating.
(Item 20)
20. The synthetic anellosome of any one of items 1-19, wherein said genetic element comprises a sequence of the consensus 5'UTR nucleic acid sequences shown in Table 16-1.
(Item 21)
21. The synthetic anellosome of any one of items 1-20, wherein said genetic element comprises the sequence of the consensus GC-rich region shown in Table 16-2.
(Item 22)
said genetic element comprises a sequence of at least 100 nucleotides in length, which is at least 70% (eg, about 70-100%, 75-95%, 80-95%, 85-95%, or 85-90%) 22. The synthetic anelosome according to any one of items 1 to 21, which is composed of G or C at positions of .
(Item 23)
23. The synthetic anelosome of any one of items 1-22, wherein said genetic element comprises the nucleic acid sequence of SEQ ID NO:120.
(Item 24)
24. The synthetic anelosome of any one of items 1-23, wherein said promoter element is exogenous to a wild-type Anellovirus.
(Item 25)
25. The synthetic anelosome of any one of items 1-24, wherein said promoter element is endogenous to a wild-type Anellovirus.
(Item 26)
the genetic element is about 1.5-2.0, 2.0-2.5, 2.5-3.0, 3.0-3.5, 3.1-3.6, 3.2- having a length of 3.7, 3.3-3.8, 3.4-3.9, 3.5-4.0, 4.0-4.5, or 4.5-5.0 kb; A synthetic anerosome according to any one of items 1-25.
(Item 27)
said synthetic anerosomes are capable of infecting, e.g., in vitro, human cells, e.g., blood cells, skin cells, muscle cells, nerve cells, adipocytes, endothelial cells, immune cells, hepatocytes, lung epithelial cells, item 27. The synthetic anerosome according to any one of 1-26.
(Item 28)
Any one of items 1-27, which is substantially non-immunogenic, e.g., does not induce a detectable and/or unwanted immune response, e.g., when detected according to the method described in Example 4 A synthetic anerosome as described in .
(Item 29)
said substantially non-immunogenic anelosomes are at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% as potent in a subject as in a control subject lacking an immune response. , 90%, 95%, or 100%.
(Item 30)
A population of at least 1000 of said anerosomes has at least about 100 copies (e.g. , 800, 900 or 1000 copies) of genetic elements into one or more human cells.
(Item 31)
Less than:
(I) Below:
(a) a promoter element;
(b) a nucleic acid sequence encoding an exogenous effector, said nucleic acid sequence being operably linked to said promoter element;
said extrinsic effector is:
(i) an antibody molecule that binds a growth factor or growth factor receptor, or a cytokine or cytokine receptor;
(ii) an enzyme or functional variant thereof;
(iii) hormones or functional variants thereof;
(iv) cytokines or functional variants thereof);
(v) a complement inhibitor;
(vi) growth factors;
(vi) growth factor inhibitors;
(vii) a clotting factor, or
(viii) modulators of STING/cGAS signaling
a nucleic acid sequence that is a secretory therapeutic agent selected from
(c) below:
a 5'UTR domain comprising the nucleic acid sequence of nucleotides 323-393 of SEQ ID NO:54, or a nucleic acid sequence at least 85% identical thereto;
a genetic element comprising;
(II) a polypeptide encoded by an ORF1 nucleic acid, e.g., a proteinaceous outer layer comprising an ORF1 molecule
In a synthetic anerosome containing
said genetic element is enclosed within said proteinaceous outer layer; and
A synthetic anerosome, wherein said synthetic anerosome is capable of delivering said genetic element to a human cell.
(Item 32)
A pharmaceutical composition comprising the synthetic anerosome according to any one of items 1-31 and a pharmaceutically acceptable carrier or excipient.
(Item 33)
33. The pharmaceutical composition of item 32, comprising at least 10 < ³ >, 10 < ⁴ >, 105, 10< ⁶ > , 10 < ⁷ >, 10 < ⁸ > or 10 < ⁹ > synthetic anerosomes.
(Item 34)
34. According to items 32 or 33, wherein the pharmaceutical composition has a predetermined ratio of particles: infectious units (e.g. <300:1, <200:1, <100:1, or <50:1). pharmaceutical composition.
(Item 35)
Less than:
(i) a first nucleic acid (eg, double-stranded or single-stranded circular DNA) comprising a sequence of said genetic element of the synthetic anellosome according to any one of items 1-34; and
(ii) an amino acid sequence selected from, for example, ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 listed in Table 16, or having at least 85% sequence identity thereto; a second nucleic acid sequence encoding one or more of the amino acid sequences having
The reaction mixture containing
(Item 36)
36. The reaction mixture of item 35, wherein said first nucleic acid and second nucleic acid are in the same nucleic acid molecule.
(Item 37)
36. The reaction mixture of item 35, wherein the first nucleic acid and the second nucleic acid are different nucleic acid molecules.
(Item 38)
36. The reaction mixture of item 35, wherein said first nucleic acid and second nucleic acid are different nucleic acid molecules, and said second nucleic acid is provided as double-stranded circular DNA.
(Item 39)
36. The reaction mixture of item 35, wherein said first and second nucleic acids are different nucleic acid molecules, and wherein said first and second nucleic acids are provided as double stranded circular DNA.
(Item 40)
38. The reaction mixture of item 37, wherein said second nucleic acid sequence is contained in a helper cell or helper virus.
(Item 41)
A method of making a synthetic anerosome, said method comprising:
a) below:
(i) a first nucleic acid molecule comprising said nucleic acid sequence of a genetic element of a synthetic anelosome according to any one of items 1-41;
(ii) an amino acid sequence selected from, for example, ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2, listed in any of Table 16, or at least 85% of a second nucleic acid molecule encoding one or more of the amino acid sequences with sequence identity;
providing a host cell comprising; and
b) incubating said host cell under conditions suitable to produce synthetic anerosomes;
A method thereby making said synthetic anerosomes.
(Item 42)
42. The method of item 41, further comprising introducing said first nucleic acid molecule and/or said second nucleic acid molecule into said cell prior to step (a).
(Item 43)
43. The method of item 42, wherein said second nucleic acid molecule is introduced into said host cell before, simultaneously with, or after said first nucleic acid molecule.
(Item 44)
43. The method of any one of items 41 or 42, wherein said second nucleic acid molecule is integrated into the genome of said host cell.
(Item 45)
45. The method of any one of items 41-44, wherein said second nucleic acid molecule is a helper (eg, the genome of a helper plasmid or helper virus).
(Item 46)
ORF2 molecule wherein the second nucleic acid molecule comprises the amino acid sequence [W/F] ^X7HX3CX1CX5H ⁽ ^SEQ ID NO:949) , ^{where Xn} is any consecutive sequence of n amino ^acids 45. The method of any one of items 41-44, wherein the method is encoded.
(Item 47)
A method of manufacturing a synthetic anerosome preparation, said method comprising:
a) a plurality of synthetic anellosomes according to any one of items 1-31, a pharmaceutical composition according to any one of items 32-34, or a reaction mixture according to any one of items 35-40 providing a;
b) optionally one of the following: contaminants as described herein, optical density measurements (e.g. OD260), particle counts (e.g. by HPLC), infectivity (e.g. particle:infectious unit ratio) evaluating said plurality for one or more; and
c) formulating said plurality of synthetic anerosomes, e.g., as a pharmaceutical composition suitable for administration to a subject, e.g., if one or more of the parameters of (b) meet a specified threshold;
A method, including
(Item 48)
Less than:
(i) a first nucleic acid molecule comprising said nucleic acid sequence of a genetic element of a synthetic anelosome according to any one of items 1-47; and
(ii) optionally an amino acid sequence selected from ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 listed in any of Table 16, or at least 85% thereof a second nucleic acid molecule encoding one or more of the amino acid sequences having the sequence identity of
host cells containing
(Item 49)
In a method of delivering exogenous effectors (e.g., therapeutic exogenous effectors) to mammalian cells, the following:
(a) providing a synthetic anerosome according to any one of items 1-48; and
(b) contacting a mammalian cell with said synthetic anerosomes;
includes;
wherein said synthetic anerosomes are capable of delivering said genetic elements to said mammalian cells;
optionally, said synthetic anerosomes are produced by introducing said genetic element into said host cell under conditions suitable for confining said genetic element within said proteinaceous outer layer in said host cell;
A method whereby said therapeutic exogenous effector is delivered to said mammalian cell.
(Item 50)
Use of a synthetic anelosome according to any one of items 1-31 or a pharmaceutical composition according to any one of items 32-34 for the delivery of said genetic element to a host cell.
(Item 51)
Use of the synthetic anerosomes according to any one of items 1-31 or the pharmaceutical composition according to any one of items 32-34 for the treatment of a disease or disorder in a subject.
(Item 52)
A synthetic anelosome according to any one of items 1-31 or a pharmaceutical composition according to any one of items 32-34 for use in treating a disease or disorder in a subject.
(Item 53)
A method of treating a disease or disorder in a subject, said method comprising administering to said subject a synthetic anerosome according to any one of items 1-31 or a pharmaceutical composition according to any one of items 32-34. A method comprising administering to
(Item 54)
Use of a synthetic anerosome according to any one of items 1-31 or a pharmaceutical composition according to any one of items 32-34 in the manufacture of a medicament for the treatment of a disease or disorder in a subject.

Claims

Less than:
(I) Below:
(a) a promoter element;
(b) a nucleic acid sequence encoding an exogenous effector, said nucleic acid sequence being operably linked to said promoter element, said exogenous effector comprising a secreted polypeptide;
(c) a sequence having at least 75% sequence identity to the 5'UTR conserved domain of wild-type Anellovirus;
or the complement thereof;
(II) A proteinaceous outer layer comprising a polypeptide encoded by an Anellovirus ORF1 nucleic acid, such as an ORF1 molecule
in a synthetic particle comprising;
said genetic element or said complement thereof is enclosed within said proteinaceous outer layer; and
A synthetic particle, wherein said synthetic particle is capable of delivering said genetic element or said complement thereof into a human cell.

said polypeptide encoded by an Anellovirus ORF1 nucleic acid comprises an ORF1 molecule, optionally:
(i) said ORF1 molecule comprises the amino acid sequence of SEQ ID NO: 217 or an amino acid sequence having at least 90% identity thereto;
(ii) the ORF1 molecule is proteolytically processed;
(iii) said ORF1 molecule is encoded by nucleotides 612-2612 of SEQ ID NO:54;
(iv) said ORF1 molecule has an amino acid sequence of an arg-rich region, jelly roll domain, hypervariable domain, N22 domain, and/or C-terminal domain listed in Table 16, or at least 85% identity thereto; and/or
(v) said ORF1 molecule comprises the amino acid sequence of SEQ ID NO: 58 or a nucleic acid sequence having at least 85% sequence identity thereto;
A synthetic particle according to claim 1 .

said genetic element or said complement thereof:
(i) the nucleic acid sequence of nucleotides 2868-2929 of SEQ ID NO:54, or a nucleic acid sequence having at least 85% sequence identity thereto; and/or
(ii) a 5'UTR domain comprising the nucleic acid sequence of nucleotides 323-393 of SEQ ID NO:54, or a nucleic acid sequence at least 90% identical thereto
3. The synthetic particle of claim 1 or 2, comprising

(i) the synthetic particle has an amino acid sequence of ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 listed in Table 16, or an amino acid sequence having at least 85% identity thereto; further comprising a polypeptide comprising; and/or
(ii) said genetic element or said complement thereof has an amino acid sequence of ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 listed in Table 16, or at least 85% thereof encoding an amino acid sequence having the identity of
Synthetic particles according to any one of claims 1-3.

(i) the synthetic particle has an amino acid sequence of ORF2, ORF2/2, ORF2/3, TAIP, ORF1/1, or ORF1/2 listed in Table 16, or at least 85% identity thereto; does not contain a polypeptide comprising an amino acid sequence; and/or
(ii) said genetic element or said complement thereof has an amino acid sequence of ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 listed in Table 16, or at least 85% thereof does not encode an amino acid sequence having the identity of
Synthetic particles according to any one of claims 1-3.

(i) the ORF1 molecule has the amino acid sequence YNPX ^２2 DXGX ^２2 N (SEQ ID NO: 829) (X ^ｎn are each independently a contiguous sequence of any n amino acids);
(ii) said ORF1 molecule has the amino acid sequence YNPX ^２2 DXGX ^２2 further comprising a first β-strand and a second β-strand flanking N (SEQ ID NO: 829), e.g., said first β-strand comprises said amino acid sequence YNPX ^２2 DXGX ^２2 N (SEQ ID NO: 829) tyrosine (Y) residues and/or said second β-strand comprises said amino acid sequence YNPX ^２2 DXGX ^２2 contains the second asparagine (N) residue (N to C) of N (SEQ ID NO: 829); and/or
(iii) the ORF1 molecule is, in order from the N-terminus to the C-terminus, the 1st β-strand, the 2nd β-strand, the 1st α-helix, the 3rd β-strand, the 4th β-strand, the 5th β-strand, the 2nd α-helix, the 6th β-strand; β-strand 7, β-strand 8, and β-strand 9,
Synthetic particles according to any one of claims 1-5.

said genetic element or said complement thereof:
(i) capable of being amplified by rolling circle replication within a host cell to produce, for example, at least 8 copies;
(ii) is single-stranded;
(iii) is cyclic;
(iv) is DNA;
(v) is negative strand DNA;
(vi) at a frequency of less than 10%, 8%, 6%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1% of said particles entering said cells incorporated, e.g., said synthetic particles are non-integral;
(vii) comprising a sequence of the consensus 5'UTR nucleic acid sequences shown in Table 38;
(viii) containing the sequences of the consensus GC-rich regions shown in Table 39;
(ix) a sequence of at least 100 nucleotides in length, which is at least 70% (eg, about 70-100%, 75-95%, 80-95%, 85-95%, or 85-90%) positioned consisting of G or C of
(x) comprises the nucleic acid sequence of SEQ ID NO: 120; and/or
(xi) about 1.5-2.0, 2.0-2.5, 2.5-3.0, 3.0-3.5, 3.1-3.6, 3.2-3. 7, 3.3-3.8, 3.4-3.9, 3.5-4.0, 4.0-4.5, or 4.5-5.0 kb in length,
Synthetic particles according to any one of claims 1-6.

8. A promoter element according to any one of claims 1 to 7, wherein the promoter element is exogenous to the wild-type Anellovirus or endogenous to the wild-type Anellovirus. synthetic particles.

(i) the synthetic particles are capable of infecting, e.g., in vitro, human cells, e.g., blood cells, skin cells, muscle cells, nerve cells, adipocytes, endothelial cells, immune cells, liver cells, lung epithelial cells; can;
(ii) said synthetic particles are substantially non-immunogenic, e.g. do not induce a detectable and/or unwanted immune response, e.g. when detected according to the method described in Example 4; Optionally, said substantially non-immunogenic particles are at least about 10%, 20%, 30%, 40%, 50%, 60%, 70% as effective in a subject as a control subject lacking an immune response. %, 80%, 90%, 95%, or 100%; and/or
(iii) a population of at least 1000 of said particles has at least about 100 copies (e.g. 600, 700, 800, 900, or 1000 copies) of the genetic element or its complement into one or more human cells;
Synthetic particles according to any one of claims 1-8.

(i) said exogenous effector is a therapeutic secreted polypeptide; and/or
(ii) said secreted polypeptide is:
an antibody molecule that binds a growth factor or growth factor receptor, or a cytokine or cytokine receptor;
an enzyme or functional variant thereof;
hormones or functional variants thereof;
cytokines or functional variants thereof);
complement inhibitors;
growth factors,
growth factor inhibitors,
a clotting factor, or
Modulator of STING/cGAS signaling
including
optionally, said antibody molecule is an anti-VEGFR antibody molecule or an anti-VEGF antibody molecule (e.g. bevacizumab, or a functional variant thereof) and/or
optionally, said antibody molecule is a scFv
Synthetic particles according to any one of claims 1-9.

A pharmaceutical composition comprising a synthetic particle according to any one of claims 1-10 and a pharmaceutically acceptable carrier or excipient.

(i) the pharmaceutical composition comprises at least 10 ^３3 , 10 ^４4 , 105, 10 ^６6 , 10 ^７7 , 10 ^８8 , or 10 ^９9 and/or
(ii) the pharmaceutical composition has a predetermined ratio of particle:infectious units (e.g., <300:1, <200:1, <100:1, or <50:1);
A pharmaceutical composition according to claim 11 .

Less than:
(i) a first nucleic acid (eg, double-stranded or single-stranded circular DNA) comprising a sequence of said genetic element or said complement thereof of the synthetic particle of any one of claims 1-10; and
(ii) an amino acid sequence selected from, for example, ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 listed in Table 16, or having at least 85% sequence identity thereto; a second nucleic acid sequence encoding one or more of the amino acid sequences having
The reaction mixture containing

(i) said first and second nucleic acids are in the same nucleic acid molecule;
(ii) said first and second nucleic acids are different nucleic acid molecules, optionally said second nucleic acid sequence is contained in a helper cell or helper virus;
(iii) said first nucleic acid and second nucleic acid are different nucleic acid molecules, and said second nucleic acid is provided as a double-stranded circular DNA; or
(iv) the first and second nucleic acids are different nucleic acid molecules, and the first and second nucleic acids are provided as double-stranded circular DNA;
14. A reaction mixture according to claim 13.

A method of making synthetic particles, said method comprising:
a) below:
(i) a first nucleic acid molecule comprising said nucleic acid sequence of a genetic element or complement thereof of the synthetic particle of any one of claims 1-10;
(ii) an amino acid sequence selected from, for example, ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2, listed in any of Table 16, or at least 85% of a second nucleic acid molecule encoding one or more of the amino acid sequences with sequence identity;
providing a host cell comprising; and
b) incubating said host cell under conditions suitable for making synthetic particles;
A method thereby making said synthetic particles.

(i) said method further comprises, prior to step (a), introducing said first nucleic acid molecule and/or said second nucleic acid molecule into said cell, optionally said second nucleic acid molecule comprising: introduced into said host cell before, simultaneously with, or after said first nucleic acid molecule;
(ii) said second nucleic acid molecule is integrated into the genome of said host cell;
(iii) said second nucleic acid molecule is a helper (eg, the genome of a helper plasmid or helper virus); and/or
(iv) the second nucleic acid molecule has the amino acid sequence [W/F]X ^７7 HX ^３3 CX ^１1 CX ^５5 H (SEQ ID NO: 949) (X ^ｎn is a contiguous sequence of any n amino acids),
16. The method of claim 15.

A method of manufacturing a synthetic particle preparation, said method comprising:
a) providing a plurality of synthetic particles according to any one of claims 1 to 10, a pharmaceutical composition according to claims 11 or 12, or a reaction mixture according to claims 13 or 14;
b) optionally one of the following: contaminants as described herein, optical density measurements (e.g. OD260), particle counts (e.g. by HPLC), infectivity (e.g. particle:infectious unit ratio) evaluating said plurality for one or more; and
c) formulating said plurality of synthetic particles, e.g., as a pharmaceutical composition suitable for administration to a subject, e.g., if one or more of the parameters of (b) meet a specified threshold;
A method, including

Less than:
(i) a first nucleic acid molecule comprising said nucleic acid sequence of the genetic element of the synthetic particle of any one of claims 1-10 or its complement, and
(ii) optionally an amino acid sequence selected from ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 listed in any of Table 16, or at least 85% thereof a second nucleic acid molecule encoding one or more of the amino acid sequences having the sequence identity of
host cells containing

An ex vivo method of delivering an exogenous effector (e.g., a therapeutic exogenous effector) to a mammalian cell, said method comprising:
(a) providing a synthetic particle according to any one of claims 1-10; and
(b) contacting mammalian cells with said synthetic particles;
includes;
wherein said synthetic particle is capable of delivering said genetic element or said complement thereof to said mammalian cell;
said secretory polypeptide is a therapeutic secretory polypeptide;
Optionally, said synthetic particle is produced by introducing said genetic element into said host cell under conditions suitable to confine said genetic element or said complement thereof within said proteinaceous outer layer in said host cell. be;
A method whereby said therapeutic exogenous effector is delivered to said mammalian cell.

A composition or pharmaceutical composition for use in treating a disease or disorder in a subject, the composition comprising the synthetic particles of any one of claims 1 to 10 or any of claims 11 or 12. The pharmaceutical composition according to item 1.

Use of a synthetic particle according to any one of claims 1-10 or a pharmaceutical composition according to claims 11 or 12 in the manufacture of a medicament for the treatment of a disease or disorder in a subject.